Dendritic enriched secreted lymphocyte activation molecule

ABSTRACT

The present invention relates to a novel human protein called Dendritic Enriched Secreted Lymphocyte Activation Molecule, and isolated polynucleotides encoding this protein. Also provided are vectors, host cells, antibodies, and recombinant methods for producing this human protein. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating disorders related to this novel human protein.

[0001] This application is a divisional of U.S. application Ser. No.09/369,248, filed Aug. 5, 1999, which is a continuation-in-part of U.S.application Ser. No. 09/244,110, filed Feb. 4, 1999, which claimsbenefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.60/073,962, filed Feb. 6, 1998, and U.S. Provisional Application No.60/078,572, filed Mar. 19, 1998, all of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to a novel human gene encoding apolypeptide which is a member of the Secreted Lymphocyte ActivationMolecule (SLAM) family. More specifically, the present invention relatesto a polynucleotide encoding a novel human polypeptide named DendriticEnriched Secreted Lymphocyte Activation Molecule, or “D-SLAM.” Thisinvention also relates to D-SLAM polypeptides, as well as vectors, hostcells, antibodies directed to D-SLAM polypeptides, and the recombinantmethods for producing the same. Also provided are diagnostic methods fordetecting disorders related to the immune system, and therapeuticmethods for treating such disorders. The invention further relates toscreening methods for identifying agonists and antagonists of D-SLAMactivity.

BACKGROUND OF THE INVENTION

[0003] A member of the immunoglobulin gene superfamily, SLAM is rapidlyinduced after activation of naive T- and B-cells. (Cocks, B. G., “ANovel Receptor Involved in T-Cell Activation,” Nature 376:260-263(1995); Aversa, G., “Engagement of the Signaling Lymphocytic ActivationMolecule (SLAM) on Activated T Cells Results in I1-2-Independent,Cyclosporin A-Sensitive T Cell Proliferation and IFN-γ Production,” J.Immun. 4036-4044 (1997).) A multifunctional 70 kDa glycoprotein , SLAMcauses proliferation and differentiation of immune cells. (Punnonen, J.,“Soluble and Membrane-bound Forms of Signaling Lymphocytic ActivationMolecule (SLAM) Induce Proliferation and Ig Synthesis by Activated HumanB Lymphocytes,” J. Exp. Med. 185:993-1004 (1997).) To elicit an immuneresponse, both a secreted form of SLAM, as well as a membrane boundedSLAM, are thought to interact.

[0004] It is also known that dendritic cells (DC) are the principalantigen presenting cells involved in primary immune responses; theirmajor function is to obtain antigen in tissues, migrate to lymphoidorgans, and activate T cells. (Mohamadzadeh, M. et al., J. Immunol. 156:3102-3106 (1996).) In fact, DC are usually the first immune cells toarrive at sites of inflammation on mucous membranes. (See, e.g.,Weissman, D. et al., J. Immunol. 155:4111-4117 (1995).) There is aconstant need to identify new polypeptide factors which may mediateinteractions between DC and T cells, leading to the activation and/orproliferation of immune cells. To date, however, SLAM molecules have notbeen identified on DC cells.

[0005] Thus, there is a need for polypeptides that affect theproliferation, activation, survival, and/or differentiation of immunecells, such as T- and B-cells, since disturbances of such regulation maybe involved in disorders relating to immune system. Therefore, there isa need for identification and characterization of such humanpolypeptides which can play a role in detecting, preventing,ameliorating or correcting such disorders.

SUMMARY OF THE INVENTION

[0006] The present invention relates to a novel polynucleotide and theencoded polypeptide of D-SLAM. Moreover, the present invention relatesto vectors, host cells, antibodies, and recombinant methods forproducing the polypeptides and polynucleotides. Also provided arediagnostic methods for detecting disorders relates to the polypeptides,and therapeutic methods for treating such disorders. The inventionfurther relates to screening methods for identifying binding partners ofD-SLAM.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIGS. 1A-1D show the nucleotide sequence (SEQ ID NO:1) and thededuced amino acid sequence (SEQ ID NO:2) of D-SLAM. The predictedleader sequence located at about amino acids 1-22 is underlined.

[0008]FIG. 2 shows the regions of identity between the amino acidsequence of the D-SLAM protein and the translation product of the humanSLAM (Accession No. gi/984969) (SEQ ID NO:3), determined by BLASTanalysis. Identical amino acids between the two polypeptides are shaded,while conservative amino acid are boxed. By examining the regions ofamino acids shaded and/or boxed, the skilled artisan can readilyidentify conserved domains between the two polypeptides. These conserveddomains are preferred embodiments of the present invention.

[0009]FIG. 3 shows an analysis of the D-SLAM amino acid sequence. Alpha,beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown, and all were generated using the defaultsettings. In the “Antigenic Index or Jameson-Wolf” graph, the positivepeaks indicate locations of the highly antigenic regions of the D-SLAMprotein, i.e., regions from which epitope-bearing peptides of theinvention can be obtained. The domains defined by these graphs arecontemplated by the present invention. Tabular representation of thedata summarized graphically in FIG. 3 can be found in Table 1.

[0010] The columns are labeled with the headings “Res”, “Position”, andRoman Numerals I-XIV. The column headings refer to the followingfeatures of the amino acid sequence presented in FIG. 3, and Table I:“Res”: amino acid residue of SEQ ID NO:2 and FIGS. 1A and 1B;“Position”: position of the corresponding residue within SEQ ID NO:2 andFIGS. 1A and 1B; I: Alpha, Regions—Garnier-Robson; II: Alpha,Regions—Chou-Fasman; III: Beta, Regions—Garnier-Robson; IV: Beta,Regions—Chou-Fasman; V: Turn, Regions—Garnier-Robson; VI: Turn,Regions—Chou-Fasman; VII: Coil, Regions—Garnier-Robson; VIII:Hydrophilicity Plot—Kyte-Doolittle; IX: Hydrophobicity Plot—Hopp-Woods;X: Alpha, Amphipathic Regions—Eisenberg; XI: Beta, AmphipathicRegions—Eisenberg; XII: Flexible Regions—Karplus-Schulz; XIII: AntigenicIndex—Jameson-Wolf; and XIV: Surface Probability Plot—Emini.

DETAILED DESCRIPTION

[0011] Definitions

[0012] The following definitions are provided to facilitateunderstanding of certain terms used throughout this specification.

[0013] In the present invention, “isolated” refers to material removedfrom its original environment (e.g., the natural environment if it isnaturally occurring), and thus is altered “by the hand of man” from itsnatural state. For example, an isolated polynucleotide could be part ofa vector or a composition of matter, or could be contained within acell, and still be “isolated” because that vector, composition ofmatter, or particular cell is not the original environment of thepolynucleotide.

[0014] In the present invention, a “secreted” D-SLAM protein refers to aprotein capable of being directed to the ER, secretory vesicles, or theextracellular space as a result of a signal sequence, as well as aD-SLAM protein released into the extracellular space without necessarilycontaining a signal sequence. If the D-SLAM secreted protein is releasedinto the extracellular space, the D-SLAM secreted protein can undergoextracellular processing to produce a “mature” D-SLAM protein. Releaseinto the extracellular space can occur by many mechanisms, includingexocytosis and proteolytic cleavage.

[0015] As used herein, a D-SLAM “polynucleotide” refers to a moleculehaving a nucleic acid sequence contained in SEQ ID NO:1 or the cDNAcontained within the clone deposited with the ATCC. For example, theD-SLAM polynucleotide can contain the nucleotide sequence of the fulllength cDNA sequence, including the 5′ and 3′ untranslated sequences,the coding region, with or without the signal sequence, the secretedprotein coding region, as well as fragments, epitopes, domains, andvariants of the nucleic acid sequence. Moreover, as used herein, aD-SLAM “polypeptide” refers to a molecule having the translated aminoacid sequence generated from the polynucleotide as broadly defined.

[0016] In specific embodiments, the polynucleotides of the invention areless than 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, or 7.5 kb inlength. In a further embodiment, polynucleotides of the inventioncomprise at least 15 contiguous nucleotides of D-SLAM coding sequence,but do not comprise all or a portion of any D-SLAM intron. In anotherembodiment, the nucleic acid comprising D-SLAM coding sequence does notcontain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ tothe D-SLAM gene in the genome).

[0017] In the present invention, the full length D-SLAM sequenceidentified as SEQ ID NO:1 was generated by overlapping sequences of thedeposited clone (contig analysis). A representative clone containing allor most of the sequence for SEQ ID NO:1 was deposited with the AmericanType Culture Collection (“ATCC”) on Feb. 6, 1998, and was given the ATCCDeposit Number 209623. The ATCC is located at 10801 UniversityBoulevard, Manassas, Va. 20110-2209, U.S.A. The ATCC deposit was madepursuant to the terms of the Budapest Treaty on the internationalrecognition of the deposit of microorganisms for purposes of patentprocedure.

[0018] A D-SLAM “polynucleotide” also includes those polynucleotidescapable of hybridizing, under stringent hybridization conditions, tosequences contained in SEQ ID NO:1, the complement thereof, or the cDNAwithin the deposited clone. “Stringent hybridization conditions” refersto an overnight incubation at 42 degree C. in a solution comprising 50%formamide, 5× SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodiumphosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20μg/ml denatured, sheared salmon sperm DNA, followed by washing thefilters in 0.1× SSC at about 65 degree C.

[0019] Also contemplated are nucleic acid molecules that hybridize tothe D-SLAM polynucleotides at moderately high stringency hybridizationconditions. Changes in the stringency of hybridization and signaldetection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example,moderately high stringency conditions include an overnight incubation at37 degree C. in a solution comprising 6× SSPE (20× SSPE=3M NaCl; 0.2MNaH₂PO₄; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmonsperm blocking DNA; followed by washes at 50 degree C. with 1× SSPE,0.1% SDS. In addition, to achieve even lower stringency, washesperformed following stringent hybridization can be done at higher saltconcentrations (e.g. 5× SSC).

[0020] Note that variations in the above conditions may be accomplishedthrough the inclusion and/or substitution of alternate blocking reagentsused to suppress background in hybridization experiments. Typicalblocking reagents include Denhardt's reagent, BLOTTO, heparin, denaturedsalmon sperm DNA, and commercially available proprietary formulations.The inclusion of specific blocking reagents may require modification ofthe hybridization conditions described above, due to problems withcompatibility.

[0021] Of course, a polynucleotide which hybridizes only to polyA+sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in thesequence listing), or to a complementary stretch of T (or U) residues,would not be included in the definition of “polynucleotide,” since sucha polynucleotide would hybridize to any nucleic acid molecule containinga poly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

[0022] The D-SLAM polynucleotide can be composed of anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. For example, D-SLAM polynucleotidescan be composed of single- and double-stranded DNA, DNA that is amixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, the D-SLAMpolynucleotides can be composed of triple-stranded regions comprisingRNA or DNA or both RNA and DNA. D-SLAM polynucleotides may also containone or more modified bases or DNA or RNA backbones modified forstability or for other reasons. “Modified” bases include, for example,tritylated bases and unusual bases such as inosine. A variety ofmodifications can be made to DNA and RNA; thus, “polynucleotide”embraces chemically, enzymatically, or metabolically modified forms.

[0023] D-SLAM polypeptides can be composed of amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain amino acids other than the 20 gene-encodedamino acids. The D-SLAM polypeptides may be modified by either naturalprocesses, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in the D-SLAM polypeptide, includingthe peptide backbone, the amino acid side-chains and the amino orcarboxyl termini. It will be appreciated that the same type ofmodification may be present in the same or varying degrees at severalsites in a given D-SLAM polypeptide. Also, a given D-SLAM polypeptidemay contain many types of modifications. D-SLAM polypeptides may bebranched, for example, as a result of ubiquitination, and they may becyclic, with or without branching. Cyclic, branched, and branched cyclicD-SLAM polypeptides may result from posttranslation natural processes ormay be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, pegylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OFPROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12(1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al.,Ann NY Acad Sci 663:48-62 (1992).)

[0024] “SEQ ID NO:1” refers to a D-SLAM polynucleotide sequence while“SEQ ID NO:2” refers to a D-SLAM polypeptide sequence.

[0025] A D-SLAM polypeptide “having biological activity” refers topolypeptides exhibiting activity similar, but not necessarily identicalto, an activity of a D-SLAM polypeptide, including mature forms, asmeasured in a particular biological assay, with or without dosedependency. In the case where dose dependency does exist, it need not beidentical to that of the D-SLAM polypeptide, but rather substantiallysimilar to the dose-dependence in a given activity as compared to theD-SLAM polypeptide (i.e., the candidate polypeptide will exhibit greateractivity or not more than about 25-fold less and, preferably, not morethan about tenfold less activity, and most preferably, not more thanabout three-fold less activity relative to the D-SLAM polypeptide.)

[0026] D-SLAM Polynucleotides and Polypeptides

[0027] Clone HDPJO39 was isolated from a dendritic cell cDNA library.This clone contains the entire coding region identified as SEQ ID NO:2.The deposited clone contains a cDNA having a total of 3220 nucleotides,which encodes a predicted open reading frame of 285 amino acid residues.(See FIGS. 1A-1D.) The open reading frame begins at an N-terminalmethionine located at nucleotide position 92, and ends at a stop codonat nucleotide position 947. The predicted molecular weight of the D-SLAMprotein should be about 34.2 kDa.

[0028] Subsequent Northern analysis also showed D-SLAM expression indendritic cells, T cell lymphoma, lymph node, spleen, thymus, smallintestine, and uterus tissues, a pattern consistent with hematopoieticspecific expression. Expression is highest in tissues involved in immunerecognition, consistent with the enriched expression in dendritic cellsand APC's. A single primary transcript of approximately 3.5-4.0 kb isobserved, with a minor transcript of 7-9 kb that likely represents anunprocessed RNA precursor. The expression of the major 3.5-4 kbtranscript is highest in lymph node, spleen, thymus, and, to a lesserdegree, in small intestine. The highest expression of the 7-9 kbtranscript is observed in the uterus.

[0029] Using BLAST analysis, SEQ ID NO:2 was found to be homologous tomembers of the Secreted Lymphocyte Activation Molecule (SLAM) family.Particularly, SEQ ID NO:2 contains domains homologous to the translationproduct of the human mRNA for SLAM (Accession No. gi/984969) (FIG. 2)(SEQ ID NO:3), including the following conserved domains: (a) apredicted transmembrane domain located at about amino acids 233-255; (b)a predicted extracellular domain located at about amino acids 23-232;and (c) a predicted intracellular domain located at about amino acids256-285. These polypeptide fragments of D-SLAM are specificallycontemplated in the present invention. Because SLAM (Accession No.gi/984969) is thought to be important in the activation andproliferation of T- and B-cells, the homology between SLAM (AccessionNo. gi/984969) and D-SLAM suggests that D-SLAM may also be involved inthe activation and proliferation of T- and B-cells.

[0030] Moreover, the encoded polypeptide has a predicted leader sequencelocated at about amino acids 1-22. (See FIGS. 1A-1D.) Also shown inFIGS. 1A-1D, the predicted secreted form of D-SLAM encompasses aboutamino acids 23-232. These polypeptide fragments of D-SLAM arespecifically contemplated in the present invention.

[0031] The D-SLAM nucleotide sequence identified as SEQ ID NO:1 wasassembled from partially homologous (“overlapping”) sequences obtainedfrom the deposited clone, and in some cases, from additional related DNAclones. The overlapping sequences were assembled into a singlecontiguous sequence of high redundancy (usually three to fiveoverlapping sequences at each nucleotide position), resulting in a finalsequence identified as SEQ ID NO:1.

[0032] Therefore, SEQ ID NO:1 and the translated SEQ ID NO:2 aresufficiently accurate and otherwise suitable for a variety of uses wellknown in the art and described further below. For instance, SEQ ID NO:1is useful for designing nucleic acid hybridization probes that willdetect nucleic acid sequences contained in SEQ ID NO:1 or the cDNAcontained in the deposited clone. These probes will also hybridize tonucleic acid molecules in biological samples, thereby enabling a varietyof forensic and diagnostic methods of the invention. Similarly,polypeptides identified from SEQ ID NO:2 may be used to generateantibodies which bind specifically to D-SLAM .

[0033] Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidescause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

[0034] Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:1 and the predicted translated amino acid sequence identified as SEQID NO:2, but also a sample of plasmid DNA containing a human cDNA ofD-SLAM deposited with the ATCC. The nucleotide sequence of the depositedD-SLAM clone can readily be determined by sequencing the deposited clonein accordance with known methods. The predicted D-SLAM amino acidsequence can then be verified from such deposits. Moreover, the aminoacid sequence of the protein encoded by the deposited clone can also bedirectly determined by peptide sequencing or by expressing the proteinin a suitable host cell containing the deposited human D-SLAM cDNA,collecting the protein, and determining its sequence.

[0035] The present invention also relates to the D-SLAM genecorresponding to SEQ ID NO:1, SEQ ID NO:2, or the deposited clone. TheD-SLAM gene can be isolated in accordance with known methods using thesequence information disclosed herein. Such methods include preparingprobes or primers from the disclosed sequence and identifying oramplifying the D-SLAM gene from appropriate sources of genomic material.

[0036] Also provided in the present invention are species homologs ofD-SLAM. Species homologs may be isolated and identified by makingsuitable probes or primers from the sequences provided herein andscreening a suitable nucleic acid source for the desired homologue.

[0037] The D-SLAM polypeptides can be prepared in any suitable manner.Such polypeptides include isolated naturally occurring polypeptides,recombinantly produced polypeptides, synthetically producedpolypeptides, or polypeptides produced by a combination of thesemethods. Means for preparing such polypeptides are well understood inthe art.

[0038] The D-SLAM polypeptides may be in the form of the secretedprotein, including the mature form, or may be a part of a largerprotein, such as a fusion protein (see below). It is often advantageousto include an additional amino acid sequence which contains secretory orleader sequences, pro-sequences, sequences which aid in purification,such as multiple histidine residues, or an additional sequence forstability during recombinant production.

[0039] D-SLAM polypeptides are preferably provided in an isolated form,and preferably are substantially purified. A recombinantly producedversion of a D-SLAM polypeptide, including the secreted polypeptide, canbe substantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988). D-SLAM polypeptides also can be purifiedfrom natural or recombinant sources using antibodies of the inventionraised against the D-SLAM protein in methods which are well known in theart.

[0040] Polynucleotide and Polypeptide Variants

[0041] “Variant” refers to a polynucleotide or polypeptide differingfrom the D-SLAM polynucleotide or polypeptide, but retaining essentialproperties thereof. Generally, variants are overall closely similar,and, in many regions, identical to the D-SLAM polynucleotide orpolypeptide.

[0042] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence of thepresent invention, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the D-SLAMpolypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may bean entire sequence shown of SEQ ID NO:1, the ORF (open reading frame),or any fragment specified as described herein.

[0043] As a practical matter, whether any particular nucleic acidmolecule or polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99%identical to a nucleotide sequence of the presence invention can bedetermined conventionally using known computer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. (1990) 6:237-245.) In a sequence alignment the query andsubject sequences are both DNA sequences. An RNA sequence can becompared by converting U's to T's. The result of said global sequencealignment is in percent identity. Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter.

[0044] If the subject sequence is shorter than the query sequencebecause of 5′ or 3′ deletions, not because of internal deletions, amanual correction must be made to the results. This is because theFASTDB program does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.Whether a nucleotide is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thiscorrected score is what is used for the purposes of the presentinvention. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score.

[0045] For example, a 90 base subject sequence is aligned to a 100 basequery sequence to determine percent identity. The deletions occur at the5′ end of the subject sequence and therefore, the FASTDB alignment doesnot show a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

[0046] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a query amino acid sequence of the presentinvention, it is intended that the amino acid sequence of the subjectpolypeptide is identical to the query sequence except that the subjectpolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the query amino acid sequence. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a query amino acid sequence, up to 5% of the amino acidresidues in the subject sequence may be inserted, deleted, (indels) orsubstituted with another amino acid. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0047] As a practical matter, whether any particular polypeptide is atleast 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequences shown in SEQ ID NO:2 or to the amino acid sequenceencoded by deposited DNA clone can be determined conventionally usingknown computer programs. A preferred method for determining the bestoverall match between a query sequence (a sequence of the presentinvention) and a subject sequence, also referred to as a global sequencealignment, can be determined using the FASTDB computer program based onthe algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245).In a sequence alignment the query and subject sequences are either bothnucleotide sequences or both amino acid sequences. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2,Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0,Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter.

[0048] If the subject sequence is shorter than the query sequence due toN- or C-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

[0049] For example, a 90 amino acid residue subject sequence is alignedwith a 100 residue query sequence to determine percent identity. Thedeletion occurs at the N-terminus of the subject sequence and therefore,the FASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

[0050] The D-SLAM variants may contain alterations in the codingregions, non-coding regions, or both. Especially preferred arepolynucleotide variants containing alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. Nucleotide variants producedby silent substitutions due to the degeneracy of the genetic code arepreferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination are also preferred.D-SLAM polynucleotide variants can be produced for a variety of reasons,e.g., to optimize codon expression for a particular host (change codonsin the human mRNA to those preferred by a bacterial host such as E.coli).

[0051] Naturally occurring D-SLAM variants are called “allelicvariants,” and refer to one of several alternate forms of a geneoccupying a given locus on a chromosome of an organism. (Genes II,Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelicvariants can vary at either the polynucleotide and/or polypeptide level.Alternatively, non-naturally occurring variants may be produced bymutagenesis techniques or by direct synthesis.

[0052] Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the D-SLAM polypeptides. For instance, one or moreamino acids can be deleted from the N-terminus or C-terminus of thesecreted protein without substantial loss of biological function. Theauthors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), reportedvariant KGF proteins having heparin binding activity even after deleting3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferongamma exhibited up to ten times higher activity after deleting 8-10amino acid residues from the carboxy terminus of this protein. (Dobeliet al., J. Biotechnology 7:199-216 (1988).)

[0053] Moreover, ample evidence demonstrates that variants often retaina biological activity similar to that of the naturally occurringprotein. For example, Gayle and coworkers (J. Biol. Chem.268:22105-22111 (1993)) conducted extensive mutational analysis of humancytokine IL-1a. They used random mutagenesis to generate over 3,500individual IL-1a mutants that averaged 2.5 amino acid changes pervariant over the entire length of the molecule. Multiple mutations wereexamined at every possible amino acid position. The investigators foundthat “[m]ost of the molecule could be altered with little effect oneither [binding or biological activity].” (See, Abstract.) In fact, only23 unique amino acid sequences, out of more than 3,500 nucleotidesequences examined, produced a protein that significantly differed inactivity from wild-type.

[0054] Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the secreted form willlikely be retained when less than the majority of the residues of thesecreted form are removed from the N-terminus or C-terminus. Whether aparticular polypeptide lacking N- or C-terminal residues of a proteinretains such immunogenic activities can readily be determined by routinemethods described herein and otherwise known in the art.

[0055] Thus, the invention further includes D-SLAM polypeptide variantswhich show substantial biological activity. Such variants includedeletions, insertions, inversions, repeats, and substitutions selectedaccording to general rules known in the art so as have little effect onactivity.

[0056] The present application is directed to nucleic acid molecules atleast 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequences disclosed herein, (e.g., encoding a polypeptide having theamino acid sequence of an N and/or C terminal deletion disclosed belowas m-n of SEQ ID NO:2), irrespective of whether they encode apolypeptide having D-SLAM functional activity. This is because evenwhere a particular nucleic acid molecule does not encode a polypeptidehaving D-SLAM functional activity, one of skill in the art would stillknow how to use the nucleic acid molecule, for instance, as ahybridization probe or a polymerase chain reaction (PCR) primer. Uses ofthe nucleic acid molecules of the present invention that do not encode apolypeptide having D-SLAM functional activity include, inter alia, (1)isolating a D-SLAM gene or allelic or splice variants thereof in a cDNAlibrary; (2) in situ hybridization (e.g., “FISH”) to metaphasechromosomal spreads to provide precise chromosomal location of theD-SLAM gene, as described in Verma et al., Human Chromosomes: A Manualof Basic Techniques, Pergamon Press, New York (1988); and (3) NorthernBlot analysis for detecting D-SLAM mRNA expression in specific tissues.

[0057] Preferred, however, are nucleic acid molecules having sequencesat least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequences disclosed herein, which do, in fact, encode a polypeptidehaving D-SLAM functional activity. By “a polypeptide having D-SLAMfunctional activity” is intended polypeptides exhibiting activitysimilar, but not necessarily identical, to a functional activity of theD-SLAM polypeptides of the present invention (e.g., complete(full-length) D-SLAM, mature D-SLAM and soluble D-SLAM (e.g., havingsequences contained in the extracellular domain of D-SLAM) as measured,for example, in a particular immunoassay or biological assay. Forexample, a D-SLAM functional activity can routinely be measured bydetermining the ability of a D-SLAM polypeptide to bind a D-SLAM ligand.D-SLAM functional activity may also be measured by determining theability of a polypeptide, such as cognate ligand which is free orexpressed on a cell surface, to induce cells expressing the polypeptide.

[0058] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 90%, 95%, 96%,97%, 98%, or 99% identical to the nucleic acid sequence of the depositedcDNA, the nucleic acid sequence shown in FIG. 1 (SEQ ID NO:1), orfragments thereof, will encode polypeptides “having D-SLAM functionalactivity.” In fact, since degenerate variants of any of these nucleotidesequences all encode the same polypeptide, in many instances, this willbe clear to the skilled artisan even without performing the abovedescribed comparison assay. It will be further recognized in the artthat, for such nucleic acid molecules that are not degenerate variants,a reasonable number will also encode a polypeptide having D-SLAMfunctional activity. This is because the skilled artisan is fully awareof amino acid substitutions that are either less likely or not likely tosignificantly effect protein function (e.g., replacing one aliphaticamino acid with a second aliphatic amino acid), as further describedbelow.

[0059] For example, guidance concerning how to make phenotypicallysilent amino acid substitutions is provided in Bowie, J. U. et al.,Science 247:1306-1310 (1990), wherein the authors indicate that thereare two main strategies for studying the tolerance of an amino acidsequence to change.

[0060] The first strategy exploits the tolerance of amino acidsubstitutions by natural selection during the process of evolution. Bycomparing amino acid sequences in different species, conserved aminoacids can be identified. These conserved amino acids are likelyimportant for protein function. In contrast, the amino acid positionswhere substitutions have been tolerated by natural selection indicatesthat these positions are not critical for protein function. Thus,positions tolerating amino acid substitution could be modified whilestill maintaining biological activity of the protein.

[0061] The second strategy uses genetic engineering to introduce aminoacid changes at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

[0062] As the authors state, these two strategies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at certain amino acid positions in the protein. For example,most buried (within the tertiary structure of the protein) amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Moreover, tolerated conservativeamino acid substitutions involve replacement of the aliphatic orhydrophobic amino acids Ala, Val, Leu and Ile; replacement of thehydroxyl residues Ser and Thr; replacement of the acidic residues Aspand Glu; replacement of the amide residues Asn and Gln, replacement ofthe basic residues Lys, Arg, and His; replacement of the aromaticresidues Phe, Tyr, and Trp, and replacement of the small-sized aminoacids Ala, Ser, Thr, Met, and Gly.

[0063] For example, site directed changes at the amino acid level ofD-SLAM can be made by replacing a particular amino acid with aconservative amino acid. Preferred conservative mutations include: M1replaced with A, G, I, L, S, T, or V; V2 replaced with A, G, I, L, S, T,or M; M3 replaced with A, G, I, L, S, T, or V; R4 replaced with H, or K;L6 replaced with A, G, I, S, T, M, or V; W7 replaced with F, or Y; S8replaced with A, G, I, T, L, T, M, or V; L9 replaced with A, G, I, S, T,M, or V; L10 replaced with A, G, I, S, T, M, or V; L11 replaced with A,G, I, S, T, M, or V; W12 replaced with F, or Y; E13 replaced with D; A14replaced with G, I, L, S, T, M, or V; L15 replaced with A, G, I, S, T,M, or V; L16 replaced with A, G, I, S, T, M, or V; I18 replaced with A,G, L, S, T, M, or V; T19 replaced with A, G, I, L, S, M, or V; V20replaced with A, G, I, L, S, T, or M; T21 replaced with A, G, I, L, S,M, or V; G22 replaced with A, I, T, L, S, T, M, or V; A23 replaced withG, I, L, S, T, M, or V; Q24 replaced with N; V25 replaced with A, G, I,T, L, S, T, or M; L26 replaced with A, G, I, S, T, M, or V; S27 replacedwith A, G, I, L, T, M, or V; K28 replaced with H, or R; V29 replacedwith A, G, I, L, S, T, or M; G30 replaced with A, I, L, S, T, M, or V;G31 replaced with A, I, L, S, T, M, or V; S32 replaced with A, G, I, L,T, M, or V; V33 replaced with A, G, I, L, S, T, or M; L34 replaced withA, G, I, S, T, M, or V; L35 replaced with A, G, I, S, T, M, or V; V36replaced with A, G, I, L, S, T, or M; A37 replaced with G, I, L, S, T,M, or V; A38 replaced with G, I, L, S, T, M, or V; R39 replaced with H,or K; G42 replaced with A, I, L, S, T, M, or V; F43 replaced with W, orY; Q44 replaced with N; V45 replaced with A, G, I, L, S, T, or M; R46replaced with H, or K; E47 replaced with D; A48 replaced with G, I, L,S, T, M, or V; I49 replaced with A, G, L, S, T, M, or V; W50 replacedwith F, or Y; R51 replaced with H, or K; S52 replaced with A, G, I, L,T, M, or V; L53 replaced with A, G, I, S, T, M, or V; W54 replaced withF, or Y; S56 replaced with A, G, I, L, T, M, or V; E57 replaced with D;E58 replaced with D; L59 replaced with A, G, I, S, T, M, or V; L60replaced with A, G, I, S, T, M, or V; A61 replaced with G, I, L, S, T,M, or V; T62 replaced with A, G, I, L, S, M, or V; F63 replaced with W,or Y; F64 replaced with W, or Y; R65 replaced with H, or K; G66 replacedwith A, I, L, S, T, M, or V; S67 replaced with A, G, I, L, T, M, or V;L68 replaced with A, G, I, S, T, M, or V; E69 replaced with D; T70replaced with A, G, I, L, S, M, or V; L71 replaced with A, G, I, S, T,M, or V; Y72 replaced with F, or W; H73 replaced with K, or R; S74replaced with A, G, I, L, T, M, or V; R75 replaced with H, or K; F76replaced with W, or Y; L77 replaced with A, G, I, S, T, M, or V; G78replaced with A, I, L, S, T, M, or V; R79 replaced with H, or K; A80replaced with G, I, L, S, T, M, or V; Q81 replaced with N; L82 replacedwith A, G, I, S, T, M, or V; H83 replaced with K, or R; S84 replacedwith A, G, I, L, T, M, or V; N85 replaced with Q; L86 replaced with A,G, I, S, T, M, or V; S87 replaced with A, G, I, L, T, M, or V; L88replaced with A, G, I, S, T, M, or V; E89 replaced with D; L90 replacedwith A, G, I, S, T, M, or V; G91 replaced with A, I, L, S, T, M, or V;L93 replaced with A, G, I, S, T, M, or V; E94 replaced with D; S95replaced with A, G, I, L, T, M, or V; G96 replaced with A, I, L, S, T,M, or V; D97 replaced with E; S98 replaced with A, G, I, L, T, M, or V;G99 replaced with A, I, L, S, T, M, or V; N100 replaced with Q; F101replaced with W, or Y; S102 replaced with A, G, I, L, T, M, or V; V103replaced with A, G, I, L, S, T, or M; L104 replaced with A, G, I, S, T,M, or V; M105 replaced with A, G, I, L, S, T, or V; V106 replaced withA, G, I, L, S, T, or M; D107 replaced with E; T108 replaced with A, G,I, L, S, M, or V; R109 replaced with H, or K; G 110 replaced with A, I,L, S, T, M, or V; Q111 replaced with N; W113 replaced with F, or Y; T114replaced with A, G, I, L, S, M, or V; Q115 replaced with N; T116replaced with A, G, I, L, S, M, or V; L117 replaced with A, G, I, S, T,M, or V; Q118 replaced with N; L119 replaced with A, G, I, S, T, M, orV; K120 replaced with H, or R; V121 replaced with A, G, I, L, S, T, orM; Y122 replaced with F, or W; D123 replaced with E; A124 replaced withG, I, L, S, T, M, or V; V125 replaced with A, G, I, L, S, T, or M; R127replaced with H, or K; V129 replaced with A, G, I, L, S, T, or M; V130replaced with A, G, I, L, S, T, or M; Q131 replaced with N; V132replaced with A, G, I, L, S, T, or M; F133 replaced with W, or Y; I134replaced with A, G, L, S, T, M, or V; A135 replaced with G, I, L, S, T,M, or V; V136 replaced with A, G, I, L, S, T, or M; E137 replaced withD; R138 replaced with H, or K; D139 replaced with E; A140 replaced withG, I, L, S, T, M, or V; Q141 replaced with N; S143 replaced with A, G,I, L, T, M, or V; K144 replaced with H, or R; T145 replaced with A, G,I, L, S, M, or V; Q147 replaced with N; V148 replaced with A, G, I, L,S, T, or M; F149 replaced with W, or Y; L150 replaced with A, G, I, S,T, M, or V; S151 replaced with A, G, I, L, T, M, or V; W153 replacedwith F, or Y; A154 replaced with G, I, L, S, T, M, or V; N156 replacedwith Q; I157 replaced with A, G, L, S, T, M, or V; S158 replaced with A,G, I, L, T, M, or V; E159 replaced with D; I160 replaced with A, G, L,S, T, M, or V; T161 replaced with A, G, I, L, S, M, or V; Y162 replacedwith F, or W; S163 replaced with A, G, I, L, T, M, or V; W164 replacedwith F, or Y; R165 replaced with H, or K; R166 replaced with H, or K;E167 replaced with D; T168 replaced with A, G, I, L, S, M, or V; T169replaced with A, G, I, L, S, M, or V; M170 replaced with A, G, I, L, S,T, or V; D171 replaced with E; F172 replaced with W, or Y; G173 replacedwith A, I, L, S, T, M, or V; M174 replaced with A, G, I, L, S, T, or V;E175 replaced with D; H177 replaced with K, or R; S178 replaced with A,G, I, L, T, M, or V; L179 replaced with A, G, I, S, T, M, or V; F180replaced with W, or Y; T181 replaced with A, G, I, L, S, M, or V; D182replaced with E; G183 replaced with A, I, L, S, T, M, or V; Q184replaced with N; V185 replaced with A, G, I, L, S, T, or M; L186replaced with A, G, I, S, T, M, or V; S 187 replaced with A, G, I, L, T,M, or V; I188 replaced with A, G, L, S, T, M, or V; S189 replaced withA, G, I, L, T, M, or V; L190 replaced with A, G, I, S, T, M, or V; G191replaced with A, I, L, S, T, M, or V; G193 replaced with A, I, L, S, T,M, or V; D194 replaced with E; R195 replaced with H, or K; D196 replacedwith E; V197 replaced with A, G, I, L, S, T, or M; A198 replaced with G,I, L, S, T, M, or V; Y199 replaced with F, or W; S200 replaced with A,G, I, L, T, M, or V; I202 replaced with A, G, L, S, T, M, or V; V203replaced with A, G, I, L, S, T, or M; S204 replaced with A, G, I, L, T,M, or V; N205 replaced with Q; V207 replaced with A, G, I, L, S, T, orM; S208 replaced with A, G, I, L, T, M, or V; W209 replaced with F, orY; D210 replaced with E; L211replaced with A, G, I, S, T, M, or V; A212replaced with G, I, L, S, T, M, or V; T213 replaced with A, G, I, L, S,M, or V; V214 replaced with A, G, I, L, S, T, or M; T215 replaced withA, G, I, L, S, M, or V; W217 replaced with F, or Y; D218 replaced withE; S219 replaced with A, G, I, L, T, M, or V; H221 replaced with K, orR; H222 replaced with K, or R; E223 replaced with D; A224 replaced withG, I, L, S, T, M, or V; A225 replaced with G, I, L, S, T, M, or V; G227replaced with A, I, L, S, T, M, or V; K228 replaced with H, or R; A229replaced with G, I, L, S, T, M, or V; S230 replaced with A, G, I, L, T,M, or V; Y231 replaced with F, or W; K232 replaced with H, or R; D233replaced with E; V234 replaced with A, G, I, L, S, T, or M; L235replaced with A, G, I, S, T, M, or V; L236 replaced with A, G, I, S, T,M, or V; V237 replaced with A, G, I, L, S, T, or M; V238 replaced withA, G, I, L, S, T, or M; V239 replaced with A, G, I, L, S, T, or M; V241replaced with A, G, I, L, S, T, or M; S242 replaced with A, G, I, L, T,M, or V; L243 replaced with A, G, I, S, T, M, or V; L244 replaced withA, G, I, S, T, M, or V; L245 replaced with A, G, I, S, T, M, or V; M246replaced with A, G, I, L, S, T, or V; L247 replaced with A, G, I, S, T,M, or V; V248 replaced with A, G, I, L, S, T, or M; T249 replaced withA, G, I, L, S, M, or V; L250 replaced with A, G, I, S, T, M, or V; F251replaced with W, or Y; S252 replaced with A, G, I, L, T, M, or V; A253replaced with G, I, L, S, T, M, or V; W254 replaced with F, or Y; H255replaced with K, or R; W256 replaced with F, or Y; S260 replaced with A,G, I, L, T, M, or V; G261 replaced with A, I, L, S, T, M, or V; K262replaced with H, or R; K263 replaced with H, or R; K264 replaced with H,or R; K265 replaced with H, or R; D266 replaced with E; V267 replacedwith A, G, I, L, S, T, or M; H268 replaced with K, or R; A269 replacedwith G, I, L, S, T, M, or V; D270 replaced with E; R271 replaced with H,or K; V272 replaced with A, G, I, L, S, T, or M; G273 replaced with A,I, L, S, T, M, or V; E275 replaced with D; T276 replaced with A, G, I,L, S, M, or V; E277 replaced with D; N278 replaced with Q; L280 replacedwith A, G, I, S, T, M, or V; V281 replaced with A, G, I, L, S, T, or M;Q282 replaced with N; D283 replaced with E; L284 replaced with A, G, I,S, T, M, or V.

[0064] The resulting constructs can be routinely screened for activitiesor functions described throughout the specification and known in theart. Preferably, the resulting constructs have an increased D-SLAMactivity or function, while the remaining D-SLAM activities or functionsare maintained. More preferably, the resulting constructs have more thanone increased D-SLAM activity or function, while the remaining D-SLAMactivities or functions are maintained.

[0065] Besides conservative amino acid substitution, variants of D-SLAMinclude (i) substitutions with one or more of the non-conserved aminoacid residues, where the substituted amino acid residues may or may notbe one encoded by the genetic code, or (ii) substitution with one ormore of amino acid residues having a substituent group, or (iii) fusionof the mature polypeptide with another compound, such as a compound toincrease the stability and/or solubility of the polypeptide (forexample, polyethylene glycol), or (iv) fusion of the polypeptide withadditional amino acids, such as an IgG Fc fusion region peptide, orleader or secretory sequence, or a sequence facilitating purification.Such variant polypeptides are deemed to be within the scope of thoseskilled in the art from the teachings herein.

[0066] For example, D-SLAM polypeptide variants containing amino acidsubstitutions of charged amino acids with other charged or neutral aminoacids may produce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

[0067] For example, preferred non-conservative substitutions of D-SLAMinclude: M1 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V2replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M3 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; R4 replaced with D, E, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; P5 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L6 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; W7 replaced with D, E, H, K, R, N, Q, A, G,I, L, S, T, M, V, P, or C; S8 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; L9 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L10replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L11 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; W12 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; E13 replaced with H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A14 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; L15 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; L16 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P17replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; I18 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T19 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V20 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; T21 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; G22 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;A23 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q24 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V25replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L26 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; S27 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; K28 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; V29 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; G30 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G31replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S32 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V33 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; L34 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; L35 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V36replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A37 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; A38 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; R39 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; P40 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or C; P41 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; G42 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; F43 replaced with D, E, H, K, R, N, Q, A, G, I, L,S, T, M, V, P, or C; Q44 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, F, W, Y, P, or C; V45 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; R46 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; E47 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; A48 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I49replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W50 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R51 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S52 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L53 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; W54 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; P55 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or C; S56 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; E57 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; E58 replaced with H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; L59 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; L60 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A61replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T62 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; F63 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; F64 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; R65 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; G66 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; S67 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; L68 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E69replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;T70 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L71 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; Y72 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; H73 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S74 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; R75 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; F76 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; L77 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; G78 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;R79 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;A80 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q81 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L82replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H83 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S84 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; N85 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; L86 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; S87 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; L88 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E89replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;L90 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G91 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; P92 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L93 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; E94 replaced with H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; S95 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; G96 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; D97 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; S98 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G99replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N100 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; F101 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S102 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V103 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; L104 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; M105 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;V106 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D107 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T108replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R109 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G110 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; Q111 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; P112 replaced with D, E, H, K,R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; W 13 replaced with D, E,H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T114 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; Q115 replaced with D, E, H, K, R, A, G,T, L, S, T, M, V, F, W, Y, P, or C; T116 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; L117 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; Q118 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,P, or C; L119 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K120replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V121replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y122 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D123 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A124 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V125 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; P126 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; R127 replaced with D, E, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; P128 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, N, Q, F, W, Y, or C; V129 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; V130 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; Q131 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; V132 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;F133 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;I134 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A135 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V136 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; E137 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; R138 replaced with D, E, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; D139 replaced with H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; A140 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; Q141 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, F, W, Y, P, or C; P142 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or C; S143 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; K144 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; T145 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;C146 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or P; Q147 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,P, or C; V148 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F149replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L150replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S151 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; C152 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; W153 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A154 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; P155 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; N156 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, F, W, Y, P, or C; I157 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; S158 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; E159 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; I160 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;T161 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y162 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S163 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; W164 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R165 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R166 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E167 replaced with H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T168 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; T169 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; M170 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; D171 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; F172 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; G173 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M174replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E175 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P176 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; H177replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S178replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L179 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; F180 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; T181 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; D182 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; G183 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; Q184 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; V185 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L186 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S187 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; I188 replaced with D, E, H,K, P, N, Q, F, W, Y, P, or C; S189 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L190 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;G191 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P192 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G193replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D194 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R195 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D196 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V197replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A198 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; Y199 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; S200 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; C201 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or P; I202 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; V203 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;S204 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N205 replacedwith D, E, H, K, R, A, G, T, L, S, T, M, V, F, W, Y, P, or C; P206replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; V207 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S208replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W209 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D210 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L211 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; A212 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; T213 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; V214 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;T215 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P216 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; W217replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D218replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;S219 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C220 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; H221replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H222replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E223replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;A224 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A225 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; P226 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G227 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; K228 replaced with D, E, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; A229 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; S230 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; Y231 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; K232 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; D233 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; V234 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L235replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L236 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V237 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; V238 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; V239 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P240replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; V241 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S242replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L243 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L244 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; L245 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; M246 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L247replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V248 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; T249 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; L250 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; F251 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; S252 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A253replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W254 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; H255 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; W256 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; C257 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; P258replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; C259 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or P; S260 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G261replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K262 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K263 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K264 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K265 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D266 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V267 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; H268 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A269 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; D270 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; R271 replaced with D, E, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; V272 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; G273 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; P274 replaced with D, E, H; K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or C; E275 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; T276 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;E277 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; N278 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; P279 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, or C; L280 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;V281 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q282 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; D283replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;L284 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P285 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C.

[0068] The resulting constructs can be routinely screened for activitiesor functions described throughout the specification and known in theart. Preferably, the resulting constructs have loss of a D-SLAM activityor function, while the remaining D-SLAM activities or functions aremaintained. More preferably, the resulting constructs have more than oneloss of D-SLAM activity or function, while the remaining D-SLAMactivities or functions are maintained.

[0069] Additionally, more than one amino acid (e.g., 2, 3, 4, 5, 6, 7,8, 9 and 10) can be replaced with the substituted amino acids asdescribed above (either conservative or nonconservative).

[0070] A further embodiment of the invention relates to a polypeptidewhich comprises the amino acid sequence of a D-SLAM polypeptide havingan amino acid sequence which contains at least one amino acidsubstitution, but not more than 50 amino acid substitutions, even morepreferably, not more than 40 amino acid substitutions, still morepreferably, not more than 30 amino acid substitutions, and still evenmore preferably, not more than 20 amino acid substitutions. Of course,in order of ever-increasing preference, it is highly preferable for apeptide or polypeptide to have an amino acid sequence which comprisesthe amino acid sequence of a D-SLAM polypeptide, which contains at leastone, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acidsubstitutions. In specific embodiments, the number of additions,substitutions, and/or deletions in the amino acid sequence of FIGS.1A-1D or fragments thereof (e.g., the mature form and/or other fragmentsdescribed herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150,conservative amino acid substitutions are preferable.

[0071] Polynucleotide and Polypeptide Fragments

[0072] The present invention is further directed to fragments of theisolated nucleic acid molecules described herein. By a fragment of anisolated nucleic acid molecule having, for example, the nucleotidesequence of the deposited cDNA (clone HDPJO39), a nucleotide sequenceencoding the polypeptide sequence encoded by the deposited cDNA, anucleotide sequence encoding the polypeptide sequence depicted in FIG. 1(SEQ ID NO:2), the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), orthe complementary strand thereto, is intended fragments at least 15 nt,and more preferably at least about 20 nt, still more preferably at least30 nt, and even more preferably, at least about 40, 50, 100, 150, 200,250, 300, 325, 350, 375, 400, 450, 500, 550, or 600 nt in length. Thesefragments have numerous uses that include, but are not limited to,diagnostic probes and primers as discussed herein. Of course, largerfragments, such as those of 501-1500 nt in length are also usefulaccording to the present invention as are fragments corresponding tomost, if not all, of the nucleotide sequences of the deposited cDNA(clone HDPJO39) or as shown in FIG. 1 (SEQ ID NO:1). By a fragment atleast 20 nt in length, for example, is intended fragments which include20 or more contiguous bases from, for example, the nucleotide sequenceof the deposited cDNA, or the nucleotide sequence as shown in FIG. 1(SEQ ID NO:1).

[0073] Moreover, representative examples of D-SLAM polynucleotidefragments include, for example, fragments having a sequence from aboutnucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300,301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750,751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100,1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400,1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700,1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, and/or2001 to the end of SEQ ID NO:1 or the complementary strand thereto, orthe cDNA contained in the deposited clone. In this context “about”includes the particularly recited ranges, larger or smaller by several(5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.

[0074] Preferably, the polynucleotide fragments of the invention encodea polypeptide which demonstrates a D-SLAM functional activity. By apolypeptide demonstrating a D-SLAM “functional activity” is meant, apolypeptide capable of displaying one or more known functionalactivities associated with a full-length (complete) D-SLAM protein. Suchfunctional activities include, but are not limited to, biologicalactivity, antigenicity [ability to bind (or compete with a D-SLAMpolypeptide for binding) to an anti-D-SLAM antibody], immunogenicity(ability to generate antibody which binds to a D-SLAM polypeptide),ability to form multimers with D-SLAM polypeptides of the invention, andability to bind to a receptor or ligand for a D-SLAM polypeptide.

[0075] The functional activity of D-SLAM polypeptides, and fragments,variants derivatives, and analogs thereof, can be assayed by variousmethods.

[0076] For example, in one embodiment where one is assaying for theability to bind or compete with full-length D-SLAM polypeptide forbinding to anti-D-SLAM antibody, various immunoassays known in the artcan be used, including but not limited to, competitive andnon-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

[0077] In another embodiment, where a D-SLAM ligand is identified, orthe ability of a polypeptide fragment, variant or derivative of theinvention to multimerize is being evaluated, binding can be assayed,e.g., by means well-known in the art, such as, for example, reducing andnon-reducing gel chromatography, protein affinity chromatography, andaffinity blotting. See generally, Phizicky, E., et al., 1995, Microbiol.Rev. 59:94-123. In another embodiment, physiological correlates ofD-SLAM binding to its substrates (signal transduction) can be assayed.

[0078] In addition, assays described herein (see Examples) and otherwiseknown in the art may routinely be applied to measure the ability ofD-SLAM polypeptides and fragments, variants derivatives and analogsthereof to elicit D-SLAM related biological activity (either in vitro orin vivo). Other methods will be known to the skilled artisan and arewithin the scope of the invention.

[0079] The present invention is further directed to fragments of theD-SLAM polypeptide described herein. By a fragment of an isolated theD-SLAM polypeptide, for example, encoded by the deposited cDNA (cloneHDPJO39), the polypeptide sequence encoded by the deposited cDNA, thepolypeptide sequence depicted in FIG. 1 (SEQ ID NO:2), is intended toencompass polypeptide fragments contained in SEQ ID NO:2 or encoded bythe cDNA contained in the deposited clone. Protein fragments may be“free-standing,” or comprised within a larger polypeptide of which thefragment forms a part or region, most preferably as a single continuousregion. Representative examples of polypeptide fragments of theinvention, include, for example, fragments from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, 161-180,181-200, 201-220, 221-240, 241-260, 261-280, or 281 to the end of thecoding region. Moreover, polypeptide fragments can be at least 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids inlength. In this context “about” includes the particularly recitedranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, ateither extreme or at both extremes.

[0080] Even if deletion of one or more amino acids from the N-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other functional activities (e.g., biologicalactivities, ability to multimerize, ability to bind D-SLAM ligand) maystill be retained. For example, the ability of shortened D-SLAM muteinsto induce and/or bind to antibodies which recognize the complete ormature forms of the polypeptides generally will be retained when lessthan the majority of the residues of the complete or mature polypeptideare removed from the N-terminus. Whether a particular polypeptidelacking N-terminal residues of a complete polypeptide retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thatan D-SLAM mutein with a large number of deleted N-terminal amino acidresidues may retain some biological or immunogenic activities. In fact,peptides composed of as few as six D-SLAM amino acid residues may oftenevoke an immune response.

[0081] Accordingly, polypeptide fragments include the secreted D-SLAMprotein as well as the mature form. Further preferred polypeptidefragments include the secreted D-SLAM protein or the mature form havinga continuous series of deleted residues from the amino or the carboxyterminus, or both. For example, any number of amino acids, ranging from1-60, can be deleted from the amino terminus of either the secretedD-SLAM polypeptide or the mature form. Similarly, any number of aminoacids, ranging from 1-30, can be deleted from the carboxy terminus ofthe secreted D-SLAM protein or mature form. Furthermore, any combinationof the above amino and carboxy terminus deletions are preferred.Similarly, polynucleotide fragments encoding these D-SLAM polypeptidefragments are also preferred.

[0082] Particularly, N-terminal deletions of the D-SLAM polypeptide canbe described by the general formula m-285, where m is an integer from 2to 284, where m corresponds to the position of the amino acid residueidentified in SEQ ID NO:2. More in particular, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, the amino acid sequence of residues of: V-2 to P-285; M-3to P-285; R-4 to P-285; P-5 to P-285; L-6 to P-285; W-7 to P-285; S-8 toP-285; L-9 to P-285; L-10 to P-285; L-11 to P-285; W-12 to P-285; E-13to P-285; A-14 to P-285; L-15 to P-285; L-16 to P-285; P-17 to P-285;I-18 to P-285; T-19 to P-285; V-20 to P-285; T-21 to P-285; G-22 toP-285; A-23 to P-285; Q-24 to P-285; V-25 to P-285; L-26 to P-285; S-27to P-285; K-28 to P-285; V-29 to P-285; G-30 to P-285; G-31 to P-285;S-32 to P-285; V-33 to P-285; L-34 to P-285; L-35 to P-285; V-36 toP-285; A-37 to P-285; A-38 to P-285; R-39 to P-285; P-40 to P-285; P-41to P-285; G-42 to P-285; F-43 to P-285; Q-44 to P-285; V-45 to P-285;R-46 to P-285; E-47 to P-285; A-48 to P-285; I-49 to P-285; W-50 toP-285; R-51 to P-285; S-52 to P-285; L-53 to P-285; W-54 to P-285; P-55to P-285; S-56 to P-285; E-57 to P-285; E-58 to P-285; L-59 to P-285;L-60 to P-285; A-61 to P-285; T-62 to P-285; F-63 to P-285; F-64 toP-285; R-65 to P-285; G-66 to P-285; S-67 to P-285; L-68 to P-285; E-69to P-285; T-70 to P-285; L-71 to P-285; Y-72 to P-285; H-73 to P-285;S-74 to P-285; R-75 to P-285; F-76 to P-285; L-77 to P-285; G-78 toP-285; R-79 to P-285; A-80 to P-285; Q-81 to P-285; L-82 to P-285; H-83to P-285; S-84 to P-285; N-85 to P-285; L-86 to P-285; S-87 to P-285;L-88 to P-285; E-89 to P-285; L-90 to P-285; G-91 to P-285; P-92 toP-285; L-93 to P-285; E-94 to P-285; S-95 to P-285; G-96 to P-285; D-97to P-285; S-98 to P-285; G-99 to P-285; N-100 to P-285; F-101 to P-285;S-102 to P-285; V-103 to P-285; L-104 to P-285; M-105 to P-285; V-106 toP-285; D-107 to P-285; T-108 to P-285; R-109 to P-285; G-110 to P-285;Q-111 to P-285; P-112 to P-285; W-113 to P-285; T-114 to P-285; Q-115 toP-285; T-116 to P-285; L-117 to P-285; Q-118 to P-285; L-119 to P-285;K-120 to P-285; V-121 to P-285; Y-122 to P-285; D-123 to P-285; A-124 toP-285; V-125 to P-285; P-126 to P-285; R-127 to P-285; P-128 to P-285;V-129 to P-285; V-130 to P-285; Q-131 to P-285; V-132 to P-285; F-133 toP-285; 1-134 to P-285; A-135 to P-285; V-136 to P-285; E-137 to P-285;R-138 to P-285; D-139 to P-285; A-140 to P-285; Q-141 to P-285; P-142 toP-285; S-143 to P-285; K-144 to P-285; T-145 to P-285; C-146 to P-285;Q-147 to P-285; V-148 to P-285; F-149 to P-285; L-150 to P-285; S-151 toP-285; C-152 to P-285; W-153 to P-285; A-154 to P-285; P-155 to P-285;N-156 to P-285; 1-157 to P-285; S-158 to P-285; E-159 to P-285; 1-160 toP-285; T-161 to P-285; Y-162 to P-285; S-163 to P-285; W-164 to P-285;R-165 to P-285; R-166 to P-285; E-167 to P-285; T-168 to P-285; T-169 toP-285; M-170 to P-285; D-171 to P-285; F-172 to P-285; G-173 to P-285;M-174 to P-285; E-175 to P-285; P-176 to P-285; H-177 to P-285; S-178 toP-285; L-179 to P-285; F-180 to P-285; T-181 to P-285; D-182 to P-285;G-183 to P-285; Q-184 to P-285; V-185 to P-285; L-186 to P-285; S-187 toP-285; 1-188 to P-285; S-189 to P-285; L-190 to P-285; G-191 to P-285;P-192 to P-285; G-193 to P-285; D-194 to P-285; R-195 to P-285; D-196 toP-285; V-197 to P-285; A-198 to P-285; Y-199 to P-285; S-200 to P-285;C-201 to P-285; 1-202 to P-285; V-203 to P-285; S-204 to P-285; N-205 toP-285; P-206 to P-285; V-207 to P-285; S-208 to P-285; W-209 to P-285;D-210 to P-285; L-211 to P-285; A-212 to P-285; T-213 to P-285; V-214 toP-285; T-215 to P-285; P-216 to P-285; W-217 to P-285; D-218 to P-285;S-219 to P-285; C-220 to P-285; H-221 to P-285; H-222 to P-285; E-223 toP-285; A-224 to P-285; A-225 to P-285; P-226 to P-285; G-227 to P-285;K-228 to P-285; A-229 to P-285; S-230 to P-285; Y-231 to P-285; K-232 toP-285; D-233 to P-285; V-234 to P-285; L-235 to P-285; L-236 to P-285;V-237 to P-285; V-238 to P-285; V-239 to P-285; P-240 to P-285; V-241 toP-285; S-242 to P-285; L-243 to P-285; L-244 to P-285; L-245 to P-285;M-246 to P-285; L-247 to P-285; V-248 to P-285; T-249 to P-285; L-250 toP-285; F-251 to P-285; S-252 to P-285; A-253 to P-285; W-254 to P-285;H-255 to P-285; W-256 to P-285; C-257 to P-285; P-258 to P-285; C-259 toP-285; S-260 to P-285; G-261 to P-285; K-262 to P-285; K-263 to P-285;K-264 to P-285; K-265 to P-285; D-266 to P-285; V-267 to P-285; H-268 toP-285; A-269 to P-285; D-270 to P-285; R-271 to P-285; V-272 to P-285;G-273 to P-285; P-274 to P-285; E-275 to P-285; T-276 to P-285; E-277 toP-285; N-278 to P-285; P-279 to P-285; L-280 to P-285; of SEQ ID NO:2.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0083] Also as mentioned above, even if deletion of one or more aminoacids from the C-terminus of a protein results in modification of lossof one or more biological functions of the protein, other functionalactivities (e.g., biological activities, ability to multimerize, abilityto bind D-SLAM ligand) may still be retained. For example the ability ofthe shortened D-SLAM mutein to induce and/or bind to antibodies whichrecognize the complete or mature forms of the polypeptide generally willbe retained when less than the majority of the residues of the completeor mature polypeptide are removed from the C-terminus. Whether aparticular polypeptide lacking C-terminal residues of a completepolypeptide retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that an D-SLAM mutein with a large number ofdeleted C-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as sixD-SLAM amino acid residues may often evoke an immune response.

[0084] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the carboxy terminus of theamino acid sequence of the D-SLAM polypeptide shown in FIG. 1 (SEQ IDNO:2), as described by the general formula 1-n, where n is an integerfrom 2 to 284, where n corresponds to the position of amino acid residueidentified in SEQ ID NO:2. More in particular, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, the amino acid sequence of residues of: M-1 to L-284; M-1to D-283; M-1 to Q-282; M-1 to V-281; M-1 to L-280; M-1 to P-279; M-1 toN-278; M-1 to E-277; M-1 to T-276; M-1 to E-275; M-1 to P-274; M-1 toG-273; M-1 to V-272; M-1 to R-271; M-1 to D-270; M-1 to A-269; M-1 toH-268; M-1 to V-267; M-1 to D-266; M-1 to K-265; M-1 to K-264; M-1 toK-263; M-1 to K-262; M-1 to G-261; M-1 to S-260; M-1 to C-259; M-1 toP-258; M-1 to C-257; M-1 to W-256; M-1 to H-255; M-1 to W-254; M-1 toA-253; M-1 to S-252; M-1 to F-251; M-1 to L-250; M-1 to T-249; M-1 toV-248; M-1 to L-247; M-1 to M-246; M-1 to L-245; M-1 to L-244; M-1 toL-243; M-1 to S-242; M-1 to V-241; M-1 to P-240; M-1 to V-239; M-1 toV-238; M-1 to V-237; M-1 to L-236; M-1 to L-235; M-1 to V-234; M-1 toD-233; M-1 to K-232; M-1 to Y-231; M-1 to S-230; M-1 to A-229; M-1 toK-228; M-1 to G-227; M-1 to P-226; M-1 to A-225; M-1 to A-224; M-1 toE-223; M-1 to H-222; M-1 to H-221; M-1 to C-220; M-1 to S-219; M-1 toD-218; M-1 to W-217; M-1 to P-216; M-1 to T-215; M-1 to V-214; M-1 toT-213; M-1 to A-212; M-1 to L-211; M-1 to D-210; M-1 to W-209; M-1 toS-208; M-I to V-207; M-1 to P-206; M-1 to N-205; M-1 to S-204; M-1 toV-203; M-1 to 1-202; M-1 to C-201; M-1 to S-200; M-1 to Y-199; M-1 toA-198; M-1 to V-197; M-1 to D-196; M-1 to R-195; M-1 to D-194; M-1 toG-193; M-1 to P-192; M-1 to G-191; M-1 to L-190; M-1 to S-189; M-1 to1-188; M-1 to S-187; M-1 to L-186; M-1 to V-185; M-1 to Q-184; M-1 toG-183; M-1 to D-182; M-1 to T-181; M-1 to F-180; M-1 to L-179; M-1 toS-178; M-1 to H-177; M-1 to P-176; M-1 to E-175; M-1 to M-174; M-1 toG-173; M-1 to F-172; M-1 to D-171; M-1 to M-170; M-1 to T-169; M-1 toT-168; M-1 to E-167; M-1 to R-166; M-1 to R-165; M-1 to W-164; M-1 toS-163; M-1 to Y-162; M-1 to T-161; M-1 to I-160; M-1 to E-159; M-1 toS-158; M-1 to I-157; M-1 to N-156; M-1 to P-155; M-1 to A-154; M-1 toW-153; M-1 to C-152; M-1 to S-151; M-1 to L-150; M-1 to F-149; M-1 toV-148; M-1 to Q-147; M-1 to C-146; M-1 to T-145; M-1 to K-144; M-1 toS-143; M-1 to P-142; M-1 to Q-141; M-1 to A-140; M-1 to D-139; M-1 toR-138; M-1 to E-137; M-1 to V-136; M-1 to A-135; M-1 to 1-134; M-1 toF-133; M-1 to V-132; M-1 to Q-131; M-1 to V-130; M-1 to V-129; M-1 toP-128; M-1 to R-127; M-1 to P-126; M-1 to V-125; M-1 to A-124; M-1 toD-123; M-1 to Y-122; M-1 to V-121; M-1 to K-120; M-1 to L-119; M-1 toQ-118; M-1 to L-117; M-1 to T-116; M-1 to Q-115; M-1 to T-114; M-1 toW-113; M-1 to P-112; M-1 to Q-111; M-1 to G-110; M-1 to R-109; M-1 toT-108; M-1 to D-107; M-1 to V-106; M-1 to M-105; M-1 to L-104; M-1 toV-103; M-1 to S-102; M-1 to F-101; M-1 to N-100; M-1 to G-99; M-1 toS-98; M-1 to D-97; M-1 to G-96; M-1 to S-95; M-1 to E-94; M-1 to L-93;M-1 to P-92; M-1 to G-91; M-1 to L-90; M-1 to E-89; M-1 to L-88; M-1 toS-87; M-1 to L-86; M-1 to N-85; M-1 to S-84; M-1 to H-83; M-1 to L-82;M-1 to Q-81; M-1 to A-80; M-1 to R-79; M-1 to G-78; M-1 to L-77; M-1 toF-76; M-1 to R-75; M-1 to S-74; M-1 to H-73; M-1 to Y-72; M-1 to L-71;M-1 to T-70; M-1 to E-69; M-1 to L-68; M-1 to S-67; M-1 to G-66; M-1 toR-65; M-1 to F-64; M-1 to F-63; M-1 to T-62; M-1 to A-61; M-1 to L-60;M-1 to L-59; M-1 to E-58; M-1 to E-57; M-1 to S-56; M-1 to P-55; M-1 toW-54; M-1 to L-53; M-1 to S-52; M-1 to R-51; M-1 to W-50; M-1 to 1-49;M-1 to A-48; M-1 to E-47; M-1 to R-46; M-1 to V-45; M-1 to Q-44; M-1 toF-43; M-1 to G-42; M-1 to P-41; M-1 to P-40; M-1 to R-39; M-1 to A-38;M-1 to A-37; M-1 to V-36; M-1 to L-35; M-1 to L-34; M-1 to V-33; M-1 toS-32; M-1 to G-31; M-1 to G-30; M-1 to V-29; M-1 to K-28; M-1 to S-27;M-1 to L-26; M-1 to V-25; M-1 to Q-24; M-1 to A-23; M-1 to G-22; M-1 toT-21; M-1 to V-20; M-1 to T-19; M-1 to I-18; M-1 to P-17; M-1 to L-16;M-1 to L-15; M-1 to A-14; M-1 to E-13; M-1 to W-12; M-1 to L-11; M-1 toL-10; M-1 to L-9; M-1 to S-8; M-1 to W-7; of SEQ ID NO:2.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0085] In addition, any of the above listed N- or C-terminal deletionscan be combined to produce a N- and C-terminal deleted D-SLAMpolypeptide. The invention also provides polypeptides having one or moreamino acids deleted from both the amino and the carboxyl termini, whichmay be described generally as having residues m-n of SEQ ID NO:2, wheren and m are integers as described above. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

[0086] Moreover, preferred N- and C-terminal deletion mutants comprise,or in the alternative consist of, the predicted secreted form of D-SLAM.Preferred secreted forms of the D-SLAM include polypeptides comprisingthe amino acid sequence of residues: M-1 to K-232; V-2 to K-232; M-3 toK-232; R-4 to K-232; P-5 to K-232; L-6 to K-232; W-7 to K-232; S-8 toK-232; L-9 to K-232; L-10 to K-232; L-11 to K-232; W-12 to K-232; E-13to K-232; A-14 to K-232; L-15 to K-232; L-16 to K-232; P-17 to K-232;1-18 to K-232; T-19 to K-232; V-20 to K-232; T-21 to K-232; G-22 toK-232; A-23 to K-232; Q-24 to K-232; V-25 to K-232; L-26 to K-232; S-27to K-232; K-28 to K-232; V-29 to K-232; G-30 to K-232; G-31 to K-232;S-32 to K-232; V-33 to K-232; L-34 to K-232; L-35 to K-232; V-36 toK-232; A-37 to K-232; A-38 to K-232; R-39 to K-232; P-40 to K-232; P-41to K-232; G-42 to K-232; F-43 to K-232; Q-44 to K-232; V-45 to K-232;R-46 to K-232; E-47 to K-232; A-48 to K-232; 1-49 to K-232; W-50 toK-232; R-51 to K-232; S-52 to K-232; L-53 to K-232; W-54 to K-232; P-55to K-232; S-56 to K-232; E-57 to K-232; E-58 to K-232; L-59 to K-232;L-60 to K-232; A-61 to K-232; T-62 to K-232; F-63 to K-232; F-64 toK-232; R-65 to K-232; G-66 to K-232; S-67 to K-232; L-68 to K-232; E-69to K-232; T-70 to K-232; L-71 to K-232; Y-72 to K-232; H-73 to K-232;S-74 to K-232; R-75 to K-232; F-76 to K-232; L-77 to K-232; G-78 toK-232; R-79 to K-232; A-80 to K-232; Q-81 to K-232; L-82 to K-232; H-83to K-232; S-84 to K-232; N-85 to K-232; L-86 to K-232; S-87 to K-232;L-88 to K-232; E-89 to K-232; L-90 to K-232; G-91 to K-232; P-92 toK-232; L-93 to K-232; E-94 to K-232; S-95 to K-232; G-96 to K-232; D-97to K-232; S-98 to K-232; G-99 to K-232; N-100 to K-232; F-101 to K-232;S-102 to K-232; V-103 to K-232; L-104 to K-232; M-105 to K-232; V-106 toK-232; D-107 to K-232; T-108 to K-232; R-109 to K-232; G-110 to K-232;Q-111 to K-232; P-112 to K-232; W-113 to K-232; T-114 to K-232; Q-115 toK-232; T-116 to K-232; L-117 to K-232; Q-118 to K-232; L-119 to K-232;K-120 to K-232; V-121 to K-232; Y-122 to K-232; D-123 to K-232; A-124 toK-232; V-125 to K-232; P-126 to K-232; R-127 to K-232; P-128 to K-232;V-129 to K-232; V-130 to K-232; Q-131 to K-232; V-132 to K-232; F-133 toK-232; I-134 to K-232; A-135 to K-232; V-136 to K-232; E-137 to K-232;R-138 to K-232; D-139 to K-232; A-140 to K-232; Q-141 to K-232; P-142 toK-232; S-143 to K-232; K-144 to K-232; T-145 to K-232; C-146 to K-232;Q-147 to K-232; V-148 to K-232; F-149 to K-232; L-150 to K-232; S-151 toK-232; C-152 to K-232; W-153 to K-232; A-154 to K-232; P-155 to K-232;N-156 to K-232; I-157 to K-232; S-158 to K-232; E-159 to K-232; I-160 toK-232; T-161 to K-232; Y-162 to K-232; S-163 to K-232; W-164 to K-232;R-165 to K-232; R-166 to K-232; E-167 to K-232; T-168 to K-232; T-169 toK-232; M-170 to K-232; D-171 to K-232; F-172 to K-232; G-173 to K-232;M-174 to K-232; E-175 to K-232; P-176 to K-232; H-177 to K-232; S-178 toK-232; L-179 to K-232; F-180 to K-232; T-181 to K-232; D-182 to K-232;G-183 to K-232; Q-184 to K-232; V-185 to K-232; L-186 to K-232; S-187 toK-232; I-188 to K-232; S-189 to K-232; L-190 to K-232; G-191 to K-232;P-192 to K-232; G-193 to K-232; D-194 to K-232; R-195 to K-232; D-196 toK-232; V-197 to K-232; A-198 to K-232; Y-199 to K-232; S-200 to K-232;C-201 to K-232; I-202 to K-232; V-203 to K-232; S-204 to K-232; N-205 toK-232; P-206 to K-232; V-207 to K-232; S-208 to K-232; W-209 to K-232;D-210 to K-232; L-211 to K-232; A-212 to K-232; T-213 to K-232; V-214 toK-232; T-215 to K-232; P-216 to K-232; W-217 to K-232; D-218 to K-232;S-219 to K-232; C-220 to K-232; H-221 to K-232; H-222 to K-232; E-223 toK-232; A-224 to K-232; A-225 to K-232; P-226 to K-232; G-227 to K-232;of SEQ ID NO:2. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0087] Additionally, preferred N- and C-terminal deletion mutantscomprise, or in the alternative consist of, fragments lacking thepredicted signal sequence of D-SLAM. Preferred fragments of D-SLAMinclude polypeptides comprising the amino acid sequence of residues:A-23 to L-284; A-23 to D-283; A-23 to Q-282; A-23 to V-281; A-23 toL-280; A-23 to P-279; A-23 to N-278; A-23 to E-277; A-23 to T-276; A-23to E-275; A-23 to P-274; A-23 to G-273; A-23 to V-272; A-23 to R-271;A-23 to D-270; A-23 to A-269; A-23 to H-268; A-23 to V-267; A-23 toD-266; A-23 to K-265; A-23 to K-264; A-23 to K-263; A-23 to K-262; A-23to G-261; A-23 to S-260; A-23 to C-259; A-23 to P-258; A-23 to C-257;A-23 to W-256; A-23 to H-255; A-23 to W-254; A-23 to A-253; A-23 toS-252; A-23 to F-251; A-23 to L-250; A-23 to T-249; A-23 to V-248; A-23to L-247; A-23 to M-246; A-23 to L-245; A-23 to L-244; A-23 to L-243;A-23 to S-242; A-23 to V-241; A-23 to P-240; A-23 to V-239; A-23 toV-238; A-23 to V-237; A-23 to L-236; A-23 to L-235; A-23 to V-234; A-23to D-233; A-23 to K-232; A-23 to Y-231; A-23 to S-230; A-23 to A-229;A-23 to K-228; A-23 to G-227; A-23 to P-226; A-23 to A-225; A-23 toA-224; A-23 to E-223; A-23 to H-222; A-23 to H-221; A-23 to C-220; A-23to S-219; A-23 to D-218; A-23 to W-217; A-23 to P-216; A-23 to T-215;A-23 to V-214; A-23 to T-213; A-23 to A-212; A-23 to L-211; A-23 toD-210; A-23 to W-209; A-23 to S-208; A-23 to V-207; A-23 to P-206; A-23to N-205; A-23 to S-204; A-23 to V-203; A-23 to 1-202; A-23 to C-201;A-23 to S-200; A-23 to Y-199; A-23 to A-198; A-23 to V-197; A-23 toD-196; A-23 to R-195; A-23 to D-194; A-23 to G-193; A-23 to P-192; A-23to G-191; A-23 to L-190; A-23 to S-189; A-23 to I-188; A-23 to S-187;A-23 to L-186; A-23 to V-185; A-23 to Q-184; A-23 to G-183; A-23 toD-182; A-23 to T-181; A-23 to F-180; A-23 to L-179; A-23 to S-178; A-23to H-177; A-23 to P-176; A-23 to E-175; A-23 to M-174; A-23 to G-173;A-23 to F-172; A-23 to D-171; A-23 to M-170; A-23 to T-169; A-23 toT-168; A-23 to E-167; A-23 to R-166; A-23 to R-165; A-23 to W-164; A-23to S-163; A-23 to Y-162; A-23 to T-161; A-23 to I-160; A-23 to E-159;A-23 to S-158; A-23 to I-157; A-23 to N-156; A-23 to P-155; A-23 toA-154; A-23 to W-153; A-23 to C-152; A-23 to S-151; A-23 to L-150; A-23to F-149; A-23 to V-148; A-23 to Q-147; A-23 to C-146; A-23 to T-145;A-23 to K-144; A-23 to S-143; A-23 to P-142; A-23 to Q-141; A-23 toA-140; A-23 to D-139; A-23 to R-138; A-23 to E-137; A-23 to V-136; A-23to A-135; A-23 to I-134; A-23 to F-133; A-23 to V-132; A-23 to Q-131;A-23 to V-130; A-23 to V-129; A-23 to P-128; A-23 to R-127; A-23 toP-126; A-23 to V-125; A-23 to A-124; A-23 to D-123; A-23 to Y-122; A-23to V-121; A-23 to K-120; A-23 to L-119; A-23 to Q-118; A-23 to L-117;A-23 to T-116; A-23 to Q-115; A-23 to T-114; A-23 to W-113; A-23 toP-112; A-23 to Q-111; A-23 to G-110; A-23 to R-109; A-23 to T-108; A-23to D-107; A-23 to V-106; A-23 to M -105; A-23 to L-104; A-23 to V-103;A-23 to S-102; A-23 to F-101; A-23 to N-100; A-23 to G-99; A-23 to S-98;A-23 to D-97; A-23 to G-96; A-23 to S-95; A-23 to E-94; A-23 to L-93;A-23 to P-92; A-23 to G-91; A-23 to L-90; A-23 to E-89; A-23 to L-88;A-23 to S-87; A-23 to L-86; A-23 to N-85; A-23 to S-84; A-23 to H-83;A-23 to L-82; A-23 to Q-81; A-23 to A-80; A-23 to R-79; A-23 to G-78;A-23 to L-77; A-23 to F-76; A-23 to R-75; A-23 to S-74; A-23 to H-73;A-23 to Y-72; A-23 to L-71; A-23 to T-70; A-23 to E-69; A-23 to L-68;A-23 to S-67; A-23 to G-66; A-23 to R-65; A-23 to F-64; A-23 to F-63;A-23 to T-62; A-23 to A-61; A-23 to L-60; A-23 to L-59; A-23 to E-58;A-23 to E-57; A-23 to S-56; A-23 to P-55; A-23 to W-54; A-23 to L-53;A-23 to S-52; A-23 to R-51; A-23 to W-50; A-23 to I-49; A-23 to A-48;A-23 to E-47; A-23 to R-46; A-23 to V-45; A-23 to Q-44; A-23 to F-43;A-23 to G-42; A-23 to P-41; A-23 to P-40; A-23 to R-39; A-23 to A-38;A-23 to A-37; A-23 to V-36; A-23 to L-35; A-23 to L-34; A-23 to V-33;A-23 to S-32; A-23 to G-31; A-23 to G-30; A-23 to V-29; of SEQ ID NO:2.

[0088] The present application is also directed to proteins containingpolypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to theD-SLAM polypeptide sequence set forth herein m-n. In preferredembodiments, the application is directed to proteins containingpolypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical topolypeptides having the amino acid sequence of the specific D-SLAM N-and C-terminal deletions recited herein. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

[0089] Among the especially preferred fragments of the invention arefragments characterized by structural or functional attributes ofD-SLAM. Such fragments include amino acid residues that comprisealpha-helix and alpha-helix forming regions (“alpha-regions”),beta-sheet and beta-sheet-forming regions (“beta-regions”), turn andturn-forming regions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, surface forming regions,and high antigenic index regions (i.e., containing four or morecontiguous amino acids having an antigenic index of greater than orequal to 1.5, as identified using the default parameters of theJameson-Wolf program) of complete (i.e., full-length) D-SLAM (SEQ IDNO:2). Certain preferred regions are those set out in FIG. 3 andinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence depicted in FIG. 1(SEQ ID NO:2), such preferred regions include; Garnier-Robson predictedalpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasmanpredicted alpha-regions, beta-regions, turn-regions, and coil-regions;Kyte-Doolittle predicted hydrophilic and hydrophobic regions; Eisenbergalpha and beta amphipathic regions; Emini surface-forming regions; andJameson-Wolf high antigenic index regions, as predicted using thedefault parameters of these computer programs. Polynucleotides encodingthese polypeptides are also encompassed by the invention.

[0090] In additional embodiments, the polynucleotides of the inventionencode functional attributes of D-SLAM. Preferred embodiments of theinvention in this regard include fragments that comprise alpha-helix andalpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheetforming regions (“beta-regions”), turn and turn-forming regions(“turn-regions”), coil and coil-forming regions (“coil-regions”),hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions andhigh antigenic index regions of D-SLAM.

[0091] The data representing the structural or functional attributes ofD-SLAM set forth in FIG. 1 and/or Table I, as described above, wasgenerated using the various modules and algorithms of the DNA*STAR seton default parameters. In a preferred embodiment, the data presented incolumns VIII, IX, XIII, and XIV of Table I can be used to determineregions of D-SLAM which exhibit a high degree of potential forantigenicity. Regions of high antigenicity are determined from the datapresented in columns VIII, IX, XIII, and/or IV by choosing values whichrepresent regions of the polypeptide which are likely to be exposed onthe surface of the polypeptide in an environment in which antigenrecognition may occur in the process of initiation of an immuneresponse.

[0092] Certain preferred regions in these regards are set out in FIG. 3,but may, as shown in Table I, be represented or identified by usingtabular representations of the data presented in FIG. 3. The DNA*STARcomputer algorithm used to generate FIG. 3 (set on the original defaultparameters) was used to present the data in FIG. 3 in a tabular format(See Table I). The tabular format of the data in FIG. 3 may be used toeasily determine specific boundaries of a preferred region.

[0093] The above-mentioned preferred regions set out in FIG. 3 and inTable I include, but are not limited to, regions of the aforementionedtypes identified by analysis of the amino acid sequence set out inFIG. 1. As set out in FIG. 3 and in Table I, such preferred regionsinclude Garnier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions,Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenbergalpha- and beta-amphipathic regions, Karplus-Schulz flexible regions,Emini surface-forming regions and Jameson-Wolf regions of high antigenicindex.

[0094] Among highly preferred fragments in this regard are those thatcomprise regions of D-SLAM that combine several structural features,such as several of the features set out in Table 1.

[0095] Other preferred fragments are biologically active D-SLAMfragments. Biologically active fragments are those exhibiting activitysimilar, but not necessarily identical, to an activity of the D-SLAMpolypeptide. The biological activity of the fragments may include animproved desired activity, or a decreased undesirable activity.

[0096] However, many polynucleotide sequences, such as EST sequences,are publicly available and accessible through sequence databases. Someof these sequences are related to SEQ ID NO:1 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. For example, the following ESTs are preferablyexcluded from the present invention: AA917335; AI094818, AI298413;N62522; AA627522; R11635; AA320408; AA379112; R09841; Z20320; N79421;D45800; T98959; AA217290; N30197; AA286132; and AA633983 (herebyincorporated by reference in their entirety.) However, to list everyrelated sequence would be cumbersome. Accordingly, preferably excludedfrom the present invention are one or more polynucleotides comprising anucleotide sequence described by the general formula of a-b, where a isany integer between 1 to 3206 of SEQ ID NO:1, b is an integer of 15 to3220, where both a and b correspond to the positions of nucleotideresidues shown in SEQ ID NO:1, and where the b is greater than or equalto a+14.

[0097] Epitope-Bearing Portions

[0098] In another aspect, the invention provides peptides andpolypeptides comprising epitope-bearing portions of the polypeptides ofthe present invention. These epitopes are immunogenic or antigenicepitopes of the polypeptides of the present invention. An “immunogenicepitope” is defined as a part of a protein that elicits an antibodyresponse in vivo when the whole polypeptide of the present invention, orfragment thereof, is the immunogen. On the other hand, a region of apolypeptide to which an antibody can bind is defined as an “antigenicdeterminant” or “antigenic epitope.” The number of in vivo immunogenicepitopes of a protein generally is less than the number of antigenicepitopes. See, e.g., Geysen, et al. (1983) Proc. Natl. Acad. Sci. USA81:3998-4002. However, antibodies can be made to any antigenic epitope,regardless of whether it is an immunogenic epitope, by using methodssuch as phage display. See e.g., Petersen G. et al. (1995) Mol. Gen.Genet. 249:425-431. Therefore, included in the present invention areboth immunogenic epitopes and antigenic epitopes.

[0099] A list of exemplified amino acid sequences comprising immunogenicepitopes are shown in Table 1 below. It is pointed out that Table 1 onlylists amino acid residues comprising epitopes predicted to have thehighest degree of antigenicity using the algorithm of Jameson and Wolf,(1988) Comp. Appl. Biosci. 4:181-186 (said references incorporated byreference in their entireties). The Jameson-Wolf antigenic analysis wasperformed using the computer program PROTEAN, using default parameters(Version 3.11 for the Power MacIntosh, DNASTAR, Inc., 1228 South ParkStreet Madison, Wis.). Table 1 and portions of polypeptides not listedin Table 1 are not considered non-immunogenic. The immunogenic epitopesof Table 1 is an exemplified list, not an exhaustive list, because otherimmunogenic epitopes are merely not recognized as such by the particularalgorithm used. Amino acid residues comprising other immunogenicepitopes may be routinely determined using algorithms similar to theJameson-Wolf analysis or by in vivo testing for an antigenic responseusing methods known in the art. See, e.g., Geysen et al., supra; U.S.Pat. Nos. 4,708,781; 5,194,392; 4,433,092; and 5,480,971 (saidreferences incorporated by reference in their entireties).

[0100] Antigenic epitope-bearing peptides and polypeptides of theinvention preferably contain a sequence of at least seven, morepreferably at least nine and most preferably between about 15 to about30 amino acids contained within the amino acid sequence of a polypeptideof the invention. Non-limiting examples of antigenic polypeptides orpeptides that can be used to D-SLAM-specific antibodies include: apolypeptide comprising amino acid residues in SEQ ID NO:2 from about29-32, 39-45, 48-50, 52-59, 64-72, 76-78, 91-101, 106-114, 121-128,136-146, 162-178, 190-198, 216-233, and 257-285. These polypeptidefragments have been determined to bear antigenic epitopes of the D-SLAMprotein by the analysis of the Jameson-Wolf antigenic index, as shown inFIG. 3, above.

[0101] It is particularly pointed out that the amino acid sequences ofTable 1 comprise immunogenic epitopes. Table 1 lists only the criticalresidues of immunogenic epitopes determined by the Jameson-Wolfanalysis. Thus, additional flanking residues on either the N-terminal,C-terminal, or both N- and C-terminal ends may be added to the sequencesof Table 1 to generate an epitope-bearing polypeptide of the presentinvention. Therefore, the immunogenic epitopes of Table 1 may includeadditional N-terminal or C-terminal amino acid residues. The additionalflanking amino acid residues may be contiguous flanking N-terminaland/or C-terminal sequences from the polypeptides of the presentinvention, heterologous polypeptide sequences, or may include bothcontiguous flanking sequences from the polypeptides of the presentinvention and heterologous polypeptide sequences.

[0102] Polypeptides of the present invention comprising immunogenic orantigenic epitopes are at least 7 amino acids residues in length. “Atleast” means that a polypeptide of the present invention comprising animmunogenic or antigenic epitope may be 7 amino acid residues in lengthor any integer between 7 amino acids and the number of amino acidresidues of the full length polypeptides of the invention. Preferredpolypeptides comprising immunogenic or antigenic epitopes are at least10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,or 100 amino acid residues in length. However, it is pointed out thateach and every integer between 7 and the number of amino acid residuesof the full length polypeptide are included in the present invention.

[0103] The immuno and antigenic epitope-bearing fragments may bespecified by either the number of contiguous amino acid residues, asdescribed above, or further specified by N-terminal and C-terminalpositions of these fragments on the amino acid sequence of SEQ ID NO:2.Every combination of a N-terminal and C-terminal position that afragment of, for example, at least 7 or at least 15 contiguous aminoacid residues in length could occupy on the amino acid sequence of SEQID NO:2 is included in the invention. Again, “at least 7 contiguousamino acid residues in length” means 7 amino acid residues in length orany integer between 7 amino acids and the number of amino acid residuesof the full length polypeptide of the present invention. Specifically,each and every integer between 7 and the number of amino acid residuesof the full length polypeptide are included in the present invention.

[0104] Immunogenic and antigenic epitope-bearing polypeptides of theinvention are useful, for example, to make antibodies which specificallybind the polypeptides of the invention, and in immunoassays to detectthe polypeptides of the present invention. The antibodies are useful,for example, in affinity purification of the polypeptides of the presentinvention. The antibodies may also routinely be used in a variety ofqualitative or quantitative immunoassays, specifically for thepolypeptides of the present invention using methods known in the art.See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press; 2nd Ed. 1988).

[0105] The epitope-bearing polypeptides of the present invention may beproduced by any conventional means for making polypeptides includingsynthetic and recombinant methods known in the art. For instance,epitope-bearing peptides may be synthesized using known methods ofchemical synthesis. For instance, Houghten has described a simple methodfor the synthesis of large numbers of peptides, such as 10-20 mgs of 248individual and distinct 13 residue peptides representing single aminoacid variants of a segment of the HA1 polypeptide, all of which wereprepared and characterized (by ELISA-type binding studies) in less thanfour weeks (Houghten, R. A. Proc. Natl. Acad. Sci. USA 82:5131-5135(1985)). This “Simultaneous Multiple Peptide Synthesis (SMPS)” processis further described in U.S. Pat. No. 4,631,211 to Houghten andcoworkers (1986). In this procedure the individual resins for thesolid-phase synthesis of various peptides are contained in separatesolvent-permeable packets, enabling the optimal use of the manyidentical repetitive steps involved in solid-phase methods. A completelymanual procedure allows 500-1000 or more syntheses to be conductedsimultaneously (Houghten et al. (1985) Proc. Natl. Acad. Sci.82:5131-5135 at 5134.

[0106] Epitope-bearing polypeptides of the present invention are used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe, et al., supra; Wilson, etal., supra, and Bittle, et al. (1985) J. Gen. Virol. 66:2347-2354. If invivo immunization is used, animals may be immunized with free peptide;however, anti-peptide antibody titer may be boosted by coupling of thepeptide to a macromolecular carrier, such as keyhole limpet hemacyanin(KLH) or tetanus toxoid. For instance, peptides containing cysteineresidues may be coupled to a carrier using a linker such as-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μgs of peptide or carrier protein and Freund's adjuvant.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibodywhich can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

[0107] As one of skill in the art will appreciate, and discussed above,the polypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to heterologous polypeptide sequences.For example, the polypeptides of the present invention may be fused withthe constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, any combination thereof including both entiredomains and portions thereof) resulting in chimeric polypeptides. Thesefusion proteins facilitate purification, and show an increased half-lifein vivo. This has been shown, e.g., for chimeric proteins consisting ofthe first two domains of the human CD4-polypeptide and various domainsof the constant regions of the heavy or light chains of mammalianimmunoglobulins. See, e.g., EPA 0,394,827; Traunecker et al. (1988)Nature 331:84-86. Fusion proteins that have a disulfide-linked dimericstructure due to the IgG portion can also be more efficient in bindingand neutralizing other molecules than monomeric polypeptides orfragments thereof alone. See, e.g., Fountoulakis et al. (1995) J.Biochem. 270:3958-3964. Nucleic acids encoding the above epitopes canalso be recombined with a gene of interest as an epitope tag to aid indetection and purification of the expressed polypeptide.

[0108] Antibodies

[0109] The present invention further relates to antibodies and T-cellantigen receptors (TCR) which specifically bind the polypeptides of thepresent invention. The antibodies of the present invention include IgG(including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2),IgD, IgE, or IgM, and IgY. As used herein, the term “antibody” (Ab) ismeant to include whole antibodies, including single-chain wholeantibodies, and antigen-binding fragments thereof. Most preferably theantibodies are human antigen binding antibody fragments of the presentinvention include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv) and fragments comprising either a V_(L) or V_(H) domain. Theantibodies may be from any animal origin including birds and mammals.Preferably, the antibodies are human, murine, rabbit, goat, guinea pig,camel, horse, or chicken.

[0110] Antigen-binding antibody fragments, including single-chainantibodies, may comprise the variable region(s) alone or in combinationwith the entire or partial of the following: hinge region, CH1, CH2, andCH3 domains. Also included in the invention are any combinations ofvariable region(s) and hinge region, CH1, CH2, and CH3 domains. Thepresent invention further includes monoclonal, polyclonal, chimeric,humanized, and human monoclonal and polyclonal antibodies whichspecifically bind the polypeptides of the present invention. The presentinvention further includes antibodies which are anti-idiotypic to theantibodies of the present invention. L1

[0111] The antibodies of the present invention may be monospecific,bispecific, trispecific or of greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of thepresent invention as well as for heterologous compositions, such as aheterologous polypeptide or solid support material. See, e.g., WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al. (1991)J. Immunol. 147:60-69; U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819,4,714,681, 4,925,648; Kostelny, S. A. et al. (1992) J. Immunol.148:1547-1553.

[0112] Antibodies of the present invention may be described or specifiedin terms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which are recognized or specifically bound by the antibody.The epitope(s) or polypeptide portion(s) may be specified as describedherein, e.g., by N-terminal and C-terminal positions, by size incontiguous amino acid residues, or listed in the Tables and Figures.Antibodies which specifically bind any epitope or polypeptide of thepresent invention may also be excluded. Therefore, the present inventionincludes antibodies that specifically bind polypeptides of the presentinvention, and allows for the exclusion of the same.

[0113] Antibodies of the present invention may also be described orspecified in terms of their cross-reactivity. Antibodies that do notbind any other analog, ortholog, or homolog of the polypeptides of thepresent invention are included. Antibodies that do not bind polypeptideswith less than 95%, less than 90%, less than 85%, less than 80%, lessthan 75%, less than 70%, less than 65%, less than 60%, less than 55%,and less than 50% identity (as calculated using methods known in the artand described herein) to a polypeptide of the present invention are alsoincluded in the present invention. Further included in the presentinvention are antibodies which only bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M,5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M,5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, and 10⁻¹⁵M.

[0114] Antibodies of the present invention have uses that include, butare not limited to, methods known in the art to purify, detect, andtarget the polypeptides of the present invention including both in vitroand in vivo diagnostic and therapeutic methods. For example, theantibodies have use in immunoassays for qualitatively and quantitativelymeasuring levels of the polypeptides of the present invention inbiological samples. See, e.g., Harlow et al., ANTIBODIES: A LABORATORYMANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988)(incorporated by reference in the entirety).

[0115] The antibodies of the present invention may be used either aloneor in combination with other compositions. The antibodies may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to polypeptides or other compositions. Forexample, antibodies of the present invention may be recombinantly fusedor conjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs, or toxins.See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 0 396 387.

[0116] The antibodies of the present invention may be prepared by anysuitable method known in the art. For example, a polypeptide of thepresent invention or an antigenic fragment thereof can be administeredto an animal in order to induce the production of sera containingpolyclonal antibodies. The term “monoclonal antibody” is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced. Monoclonal antibodies can be preparedusing a wide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technology.

[0117] Hybridoma techniques include those known in the art and taught inHarlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring HarborLaboratory Press, 2nd ed. 1988); Hammerling, et al., in: MONOCLONALANTIBODIES AND T-CELL HYBRIDOMAS 563-681 (Elsevier, N.Y., 1981) (saidreferences incorporated by reference in their entireties). Fab andF(ab′)2 fragments may be produced by proteolytic cleavage, using enzymessuch as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2fragments).

[0118] Alternatively, antibodies of the present invention can beproduced through the application of recombinant DNA and phage displaytechnology or through synthetic chemistry using methods known in theart. For example, the antibodies of the present invention can beprepared using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of a phage particle which carries polynucleotide sequencesencoding them. Phage with a desired binding property are selected from arepertoire or combinatorial antibody library (e.g. human or murine) byselecting directly with antigen, typically antigen bound or captured toa solid surface or bead. Phage used in these methods are typicallyfilamentous phage including fd and M13 with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman U. et al. (1995) J. Immunol. Methods182:41-50; Ames, R. S. et al. (1995) J. Immunol. Methods 184:177-186;Kettleborough, C. A. et al. (1994) Eur. J. Immunol. 24:952-958; Persic,L. et al. (1997) Gene 187 9-18; Burton, D. R. et al. (1994) Advances inImmunology 57:191-280; PCT/GB91/01134; WO 90/02809; WO 91/10737; WO92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S.Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908,5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225,5,658,727 and 5,733,743 (said references incorporated by reference intheir entireties).

[0119] As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired hostincluding mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′ andF(ab′)2 fragments can also be employed using methods known in the artsuch as those disclosed in WO 92/22324; Mullinax, R. L. et al. (1992)BioTechniques 12(6):864-869; and Sawai, H. et al. (1995) AJRI 34:26-34;and Better, M. et al. (1988) Science 240:1041-1043 (said referencesincorporated by reference in their entireties).

[0120] Examples of techniques which can be used to produce single-chainFvs and antibodies include those described in U.S. Pat. Nos. 4,946,778and 5,258,498; Huston et al. (1991) Methods in Enzymology 203:46-88;Shu, L. et al. (1993) PNAS 90:7995-7999; and Skerra, A. et al. (1988)Science 240:1038-1040. For some uses, including in vivo use ofantibodies in humans and in vitro detection assays, it may be preferableto use chimeric, humanized, or human antibodies. Methods for producingchimeric antibodies are known in the art. See e.g., Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies, S. D.et al. (1989) J. Immunol. Methods 125:191-202; and U.S. Pat. No.5,807,715. Antibodies can be humanized using a variety of techniquesincluding CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos.5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0519 596; Padlan E. A., (1991) Molecular Immunology 28(4/5):489-498;Studnicka G. M. et al. (1994) Protein Engineering 7(6):805-814; RoguskaM. A. et al. (1994) PNAS 91:969-973), and chain shuffling (U.S. Pat. No.5,565,332). Human antibodies can be made by a variety of methods knownin the art including phage display methods described above. See also,U.S. Pat. Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741 (said references incorporated by reference intheir entireties).

[0121] Further included in the present invention are antibodiesrecombinantly fused or chemically conjugated (including both covalentlyand non-covalently conjugations) to a polypeptide of the presentinvention. The antibodies may be specific for antigens other thanpolypeptides of the present invention. For example, antibodies may beused to target the polypeptides of the present invention to particularcell types, either in vitro or in vivo, by fusing or conjugating thepolypeptides of the present invention to antibodies specific forparticular cell surface receptors. Antibodies fused or conjugated to thepolypeptides of the present invention may also be used in in vitroimmunoassays and purification methods using methods known in the art.See e.g., Harbor et al. supra and WO 93/21232; EP 0 439 095; Naramura,M. et al. (1994) Immunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981;Gillies, S. O. et al. (1992) PNAS 89:1428-1432; Fell, H. P. et al.(1991) J. Immunol. 146:2446-2452 (said references incorporated byreference in their entireties).

[0122] The present invention further includes compositions comprisingthe polypeptides of the present invention fused or conjugated toantibody domains other than the variable regions. For example, thepolypeptides of the present invention may be fused or conjugated to anantibody Fc region, or portion thereof. The antibody portion fused to apolypeptide of the present invention may comprise the hinge region, CH1domain, CH2 domain, and CH3 domain or any combination of whole domainsor portions thereof. The polypeptides of the present invention may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046,5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367 166; WO96/04388, WO 91/06570; Ashkenazi, A. et al. (1991) PNAS 88:10535-10539;Zheng, X. X. et al. (1995) J. Immunol. 154:5590-5600; and Vil, H. et al.(1992) PNAS 89:11337-11341 (said references incorporated by reference intheir entireties).

[0123] The invention further relates to antibodies that act as agonistsor antagonists of the polypeptides of the present invention. Antibodieswhich act as agonists or antagonists of the polypeptides of the presentinvention include, for example, antibodies which disrupt receptor/ligandinteractions with the polypeptides of the invention either partially orfully. For example, the present invention includes antibodies thatdisrupt the ability of the proteins of the invention to multimerize. Inanother example, the present invention includes antibodies which allowthe proteins of the invention to multimerize, but disrupts the abilityof the proteins of the invention to bind one or more D-SLAMreceptor(s)/ligand(s). In yet another example, the present inventionincludes antibodies which allow the proteins of the invention tomultimerize, and bind D-SLAM receptor(s)/ligand(s), but blocksbiological activity associated with the D-SLAM/receptor/ligand complex.

[0124] Antibodies which act as agonists or antagonists of thepolypeptides of the present invention also include, bothreceptor-specific antibodies and ligand-specific antibodies. Includedare receptor-specific antibodies that do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. Also included are receptor-specific antibodies which both preventligand binding and receptor activation. Likewise, included areneutralizing antibodies which bind the ligand and prevent binding of theligand to the receptor, as well as antibodies which bind the ligand,thereby preventing receptor activation, but do not prevent the ligandfrom binding the receptor. Further included are antibodies that activatethe receptor. These antibodies may act as agonists for either all orless than all of the biological activities affected by ligand-mediatedreceptor activation. The antibodies may be specified as agonists orantagonists for biological activities comprising specific activitiesdisclosed herein. The above antibody agonists can be made using methodsknown in the art. See e.g., WO 96/40281; U.S. Pat. No. 5,811,097; Deng,B. et al., Blood 92(6):1981-1988 (1998); Chen, Z. et al., Cancer Res.58(16):3668-3678 (1998); Harrop, J. A. et al., J. Immunol.161(4):1786-1794 (1998); Zhu, Z. et al., Cancer Res. 58(15):3209-3214(1998); Yoon, D. Y. et al., J. Immunol. 160(7):3170-3179 (1998); Prat,M. et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard, V. et al., J.Immunol. Methods 205(2):177-190 (1997); Liautard, J. et al., Cytokinde9(4):233-241 (1997); Carlson, N. G. et al., J. Biol. Chem.272(17):11295-11301 (1997); Taryman, R. E. et al., Neuron 14(4):755-762(1995); Muller, Y. A. et al., Structure 6(9):1153-1167 (1998); Bartunek,P. et al., Cytokine 8(1):14-20 (1996) (said references incorporated byreference in their entireties).

[0125] As discussed above, antibodies to the D-SLAM proteins of theinvention can, in turn, be utilized to generate anti-idiotype antibodiesthat “mimic” D-SLAM using techniques well known to those skilled in theart. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) andNissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodieswhich bind to D-SLAM and competitively inhibit D-SLAM multimerizationand/or binding to ligand can be used to generate anti-idiotypes that“mimic” the D-SLAM mutimerization and/or binding domain and, as aconsequence, bind to and neutralize D-SLAM and/or its ligand. Suchneutralizing anti-idiotypes or Fab fragments of such anti-idiotypes canbe used in therapeutic regimens to neutralize D-SLAM ligand. Forexample, such anti-idiotypic antibodies can be used to bind D-SLAM, orto bind D-SLAM ligands/receptors, and thereby block D-SLAM biologicalactivity.

[0126] Fusion Proteins

[0127] Any D-SLAM polypeptide can be used to generate fusion proteins.For example, the D-SLAM polypeptide, when fused to a second protein, canbe used as an antigenic tag. Antibodies raised against the D-SLAMpolypeptide can be used to indirectly detect the second protein bybinding to the D-SLAM. Moreover, because secreted proteins targetcellular locations based on trafficking signals, the D-SLAM polypeptidescan be used as a targeting molecule once fused to other proteins.

[0128] Examples of domains that can be fused to D-SLAM polypeptidesinclude not only heterologous signal sequences, but also otherheterologous functional regions. The fusion does not necessarily need tobe direct, but may occur through linker sequences.

[0129] In certain preferred embodiments, D-SLAM proteins of theinvention comprise fusion proteins wherein the D-SLAM polypeptides arethose described above as m-n. In preferred embodiments, the applicationis directed to nucleic acid molecules at least 90%, 95%, 96%, 97%, 98%or 99% identical to the nucleic acid sequences encoding polypeptideshaving the amino acid sequence of the specific N- and C-terminaldeletions recited herein. Polynucleotides encoding these polypeptidesare also encompassed by the invention.

[0130] Moreover, fusion proteins may also be engineered to improvecharacteristics of the D-SLAM polypeptide. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the D-SLAM polypeptide to improve stability andpersistence during purification from the host cell or subsequenthandling and storage. Also, peptide moieties may be added to the D-SLAMpolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the D-SLAM polypeptide. The addition ofpeptide moieties to facilitate handling of polypeptides are familiar androutine techniques in the art.

[0131] Moreover, D-SLAM polypeptides, including fragments, andspecifically epitopes, can be combined with parts of the constant domainof immunoglobulins (IgG), resulting in chimeric polypeptides. Thesefusion proteins facilitate purification and show an increased half-lifein vivo. One reported example describes chimeric proteins consisting ofthe first two domains of the human CD4-polypeptide and various domainsof the constant regions of the heavy or light chains of mammalianimmunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86(1988).) Fusion proteins having disulfide-linked dimeric structures (dueto the IgG) can also be more efficient in binding and neutralizing othermolecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)

[0132] Similarly, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

[0133] Moreover, the D-SLAM polypeptides can be fused to markersequences, such as a peptide which facilitates purification of D-SLAM.In preferred embodiments, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824(1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Another peptide tag useful for purification, the “HA” tag,corresponds to an epitope derived from the influenza hemagglutininprotein. (Wilson et al., Cell 37:767 (1984).)

[0134] Thus, any of these above fusions can be engineered using theD-SLAM polynucleotides or the polypeptides.

[0135] Vectors, Host Cells, and Protein Production

[0136] The present invention also relates to vectors containing theD-SLAM polynucleotide, host cells, and the production of polypeptides byrecombinant techniques. The vector may be, for example, a phage,plasmid, viral, or retroviral vector. Retroviral vectors may bereplication competent or replication defective. In the latter case,viral propagation generally will occur only in complementing host cells.

[0137] D-SLAM polynucleotides may be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it maybe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

[0138] The D-SLAM polynucleotide insert should be operatively linked toan appropriate promoter, such as the phage lambda PL promoter, the E.coli lac, trp, phoA and tac promoters, the SV40 early and late promotersand promoters of retroviral LTRs, to name a few. Other suitablepromoters will be known to the skilled artisan. The expressionconstructs will further contain sites for transcription initiation,termination, and, in the transcribed region, a ribosome binding site fortranslation. The coding portion of the transcripts expressed by theconstructs will preferably include a translation initiating codon at thebeginning and a termination codon (UAA, UGA or UAG) appropriatelypositioned at the end of the polypeptide to be translated.

[0139] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase, G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

[0140] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Other suitable vectors will be readily apparent to the skilled artisan.

[0141] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that D-SLAM polypeptidesmay in fact be expressed by a host cell lacking a recombinant vector.

[0142] D-SLAM polypeptides can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

[0143] D-SLAM polypeptides, and preferably the secreted form, can alsobe recovered from: products purified from natural sources, includingbodily fluids, tissues and cells, whether directly isolated or cultured;products of chemical synthetic procedures; and products produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, bacterial, yeast, higher plant, insect, and mammaliancells. Depending upon the host employed in a recombinant productionprocedure, the D-SLAM polypeptides may be glycosylated or may benon-glycosylated. In addition, D-SLAM polypeptides may also include aninitial modified methionine residue, in some cases as a result ofhost-mediated processes. Thus, it is well known in the art that theN-terminal methionine encoded by the translation initiation codongenerally is removed with high efficiency from any protein aftertranslation in all eukaryotic cells. While the N-terminal methionine onmost proteins also is efficiently removed in most prokaryotes, for someproteins, this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked.

[0144] In addition to encompassing host cells containing the vectorconstructs discussed herein, the invention also encompasses primary,secondary, and immortalized host cells of vertebrate origin,particularly mammalian origin, that have been engineered to delete orreplace endogenous genetic material (e.g., D-SLAM coding sequence),and/or to include genetic material (e.g., heterologous polynucleotidesequences) that is operably associated with D-SLAM polynucleotides ofthe invention, and which activates, alters, and/or amplifies endogenousD-SLAM polynucleotides. For example, techniques known in the art may beused to operably associate heterologous control regions (e.g., promoterand/or enhancer) and endogenous D-SLAM polynucleotide sequences viahomologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issuedJun. 24, 1997; International Publication No. WO 96/29411, published Sep.26, 1996; International Publication No. WO 94/12650, published Aug. 4,1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); andZijlstra et al., Nature 342:435-438 (1989), the disclosures of each ofwhich are incorporated by reference in their entireties).

[0145] In addition, polypeptides of the invention can be chemicallysynthesized using techniques known in the art (e.g., see Creighton,1983, Proteins: Structures and Molecular Principles, W. H. Freeman &Co., N.Y., and Hunkapiller, M., et al., 1984, Nature 310:105-111). Forexample, a peptide corresponding to a fragment of the D-SLAMpolypeptides of the invention can be synthesized by use of a peptidesynthesizer. Furthermore, if desired, nonclassical amino acids orchemical amino acid analogs can be introduced as a substitution oraddition into the D-SLAM polynucleotide sequence. Non-classical aminoacids include, but are not limited to, to the D-isomers of the commonamino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid,4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids,designer amino acids such as b-methyl amino acids, Ca-methyl aminoacids, Na-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

[0146] The invention encompasses D-SLAM polypeptides which aredifferentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques, includingbut not limited, to specific chemical cleavage by cyanogen bromide,trypsin, chymotrypsin, papain, V8 protease, NaBH₄; acetylation,formylation, oxidation, reduction; metabolic synthesis in the presenceof tunicamycin; etc.

[0147] Additional post-translational modifications encompassed by theinvention include, for example, e.g., N-linked or O-linked carbohydratechains, processing of N-terminal or C-terminal ends), attachment ofchemical moieties to the amino acid backbone, chemical modifications ofN-linked or O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

[0148] Also provided by the invention are chemically modifiedderivatives of D-SLAM which may provide additional advantages such asincreased solubility, stability and circulating time of the polypeptide,or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemicalmoieties for derivitization may be selected from water soluble polymerssuch as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. The polypeptides may be modified at random positions within themolecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

[0149] The polymer may be of any molecular weight, and may be branchedor unbranched. For polyethylene glycol, the preferred molecular weightis between about 1 kDa and about 100 kDa (the term “about” indicatingthat in preparations of polyethylene glycol, some molecules will weighmore, some less, than the stated molecular weight) for ease in handlingand manufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

[0150] The polyethylene glycol molecules (or other chemical moieties)should be attached to the protein with consideration of effects onfunctional or antigenic domains of the protein. There are a number ofattachment methods available to those skilled in the art, e.g., EP 0 401384, herein incorporated by reference (coupling PEG to G-CSF), see alsoMalik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

[0151] One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated-protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

[0152] The D-SLAM polypeptides of the invention may be in monomers ormultimers (i.e., dimers, trimers, tetramers and higher multimers).Accordingly, the present invention relates to monomers and multimers ofthe D-SLAM polypeptides of the invention, their preparation, andcompositions (preferably, pharmaceutical compositions) containing them.In specific embodiments, the polypeptides of the invention are monomers,dimers, trimers or tetramers. In additional embodiments, the multimersof the invention are at least dimers, at least trimers, or at leasttetramers.

[0153] Multimers encompassed by the invention may be homomers orheteromers. As used herein, the term homomer, refers to a multimercontaining only D-SLAM polypeptides of the invention (including D-SLAMfragments, variants, splice variants, and fusion proteins, as describedherein). These homomers may contain D-SLAM polypeptides having identicalor different amino acid sequences. In a specific embodiment, a homomerof the invention is a multimer containing only D-SLAM polypeptideshaving an identical amino acid sequence. In another specific embodiment,a homomer of the invention is a multimer containing D-SLAM polypeptideshaving different amino acid sequences. In specific embodiments, themultimer of the invention is a homodimer (e.g., containing D-SLAMpolypeptides having identical or different amino acid sequences) or ahomotrimer (e.g., containing D-SLAM polypeptides having identical and/ordifferent amino acid sequences). In additional embodiments, thehomomeric multimer of the invention is at least a homodimer, at least ahomotrimer, or at least a homotetramer.

[0154] As used herein, the term heteromer refers to a multimercontaining one or more heterologous polypeptides (i.e., polypeptides ofdifferent proteins) in addition to the D-SLAM polypeptides of theinvention. In a specific embodiment, the multimer of the invention is aheterodimer, a heterotrimer, or a heterotetramer. In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

[0155] Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the D-SLAM polypeptides of theinvention. Such covalent associations may involve one or more amino acidresidues contained in the polypeptide sequence (e.g., that recited inSEQ ID NO:2, or contained in the polypeptide encoded by the cloneHDPJO39). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a D-SLAM fusion protein. In oneexample, covalent associations are between the heterologous sequencecontained in a fusion protein of the invention (see, e.g., U.S. Pat. No.5,478,925). In a specific example, the covalent associations are betweenthe heterologous sequence contained in a D-SLAM-Fc fusion protein of theinvention (as described herein). In another specific example, covalentassociations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another Secreted LymphocyteActivation Molecule (SLAM) family member that is capable of formingcovalently associated multimers, such as for example, oseteoprotegerin(see, e.g., International Publication No. WO 98/49305, the contents ofwhich are herein incorporated by reference in its entirety).

[0156] The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

[0157] Alternatively, multimers of the invention may be generated usinggenetic engineering techniques known in the art. In one embodiment,polypeptides contained in multimers of the invention are producedrecombinantly using fusion protein technology described herein orotherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety). In a specificembodiment, polynucleotides coding for a homodimer of the invention aregenerated by ligating a polynucleotide sequence encoding a polypeptideof the invention to a sequence encoding a linker polypeptide and thenfurther to a synthetic polynucleotide encoding the translated product ofthe polypeptide in the reverse orientation from the original C-terminusto the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat.No. 5,478,925, which is herein incorporated by reference in itsentirety). In another embodiment, recombinant techniques describedherein or otherwise known in the art are applied to generate recombinantpolypeptides of the invention which contain a transmembrane domain (orhyrophobic or signal peptide) and which can be incorporated by membranereconstitution techniques into liposomes (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).

[0158] Uses of the D-SLAM Polynucleotides

[0159] The D-SLAM polynucleotides identified herein can be used innumerous ways as reagents. The following description should beconsidered exemplary and utilizes known techniques.

[0160] There exists an ongoing need to identify new chromosome markers,since few chromosome marking reagents, based on actual sequence data(repeat polymorphisms), are presently available.

[0161] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the sequences shown in SEQ ID NO: 1.Primers can be selected using computer analysis so that primers do notspan more than one predicted exon in the genomic DNA. These primers arethen used for PCR screening of somatic cell hybrids containingindividual human chromosomes. Only those hybrids containing the humanD-SLAM gene corresponding to the SEQ ID NO:1 will yield an amplifiedfragment.

[0162] Similarly, somatic hybrids provide a rapid method of PCR mappingthe polynucleotides to particular chromosomes. Three or more clones canbe assigned per day using a single thermal cycler. Moreover,sublocalization of the D-SLAM polynucleotides can be achieved withpanels of specific chromosome fragments. Other gene mapping strategiesthat can be used include in situ hybridization, prescreening withlabeled flow-sorted chromosomes, and preselection by hybridization toconstruct chromosome specific-cDNA libraries.

[0163] Precise chromosomal location of the D-SLAM polynucleotides canalso be achieved using fluorescence in situ hybridization (FISH) of ametaphase chromosomal spread. This technique uses polynucleotides asshort as 500 or 600 bases; however, polynucleotides 2,000-4,000 bp arepreferred. For a review of this technique, see Verma et al., “HumanChromosomes: a Manual of Basic Techniques,” Pergamon Press, New York(1988).

[0164] For chromosome mapping, the D-SLAM polynucleotides can be usedindividually (to mark a single chromosome or a single site on thatchromosome) or in panels (for marking multiple sites and/or multiplechromosomes). Preferred polynucleotides correspond to the noncodingregions of the cDNAs because the coding sequences are more likelyconserved within gene families, thus increasing the chance of crosshybridization during chromosomal mapping.

[0165] Once a polynucleotide has been mapped to a precise chromosomallocation, the physical position of the polynucleotide can be used inlinkage analysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. (Diseasemapping data are found, for example, in V. McKusick, MendelianInheritance in Man (available on line through Johns Hopkins UniversityWelch Medical Library).) Assuming 1 megabase mapping resolution and onegene per 20 kb, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of 50-500 potential causativegenes.

[0166] Thus, once coinheritance is established, differences in theD-SLAM polynucleotide and the corresponding gene between affected andunaffected individuals can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected individuals, but not in normalindividuals, indicates that the mutation may cause the disease. However,complete sequencing of the D-SLAM polypeptide and the corresponding genefrom several normal individuals is required to distinguish the mutationfrom a polymorphism. If a new polymorphism is identified, thispolymorphic polypeptide can be used for further linkage analysis.

[0167] Furthermore, increased or decreased expression of the gene inaffected individuals as compared to unaffected individuals can beassessed using D-SLAM polynucleotides. Any of these alterations (alteredexpression, chromosomal rearrangement, or mutation) can be used as adiagnostic or prognostic marker.

[0168] In addition to the foregoing, a D-SLAM polynucleotide can be usedto control gene expression through triple helix formation or antisenseDNA or RNA. Both methods rely on binding of the polynucleotide to DNA orRNA. For these techniques, preferred polynucleotides are usually 20 to40 bases in length and complementary to either the region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. Acids Res.6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) orto the mRNA itself(antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. Both techniques are effective in model systems, andthe information disclosed herein can be used to design antisense ortriple helix polynucleotides in an effort to treat disease.

[0169] D-SLAM polynucleotides are also useful in gene therapy. One goalof gene therapy is to insert a normal gene into an organism having adefective gene, in an effort to correct the genetic defect. D-SLAMoffers a means of targeting such genetic defects in a highly accuratemanner. Another goal is to insert a new gene that was not present in thehost genome, thereby producing a new trait in the host cell.

[0170] The D-SLAM polynucleotides are also useful for identifyingindividuals from minute biological samples. The United States military,for example, is considering the use of restriction fragment lengthpolymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identifying personnel. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The D-SLAM polynucleotides can beused as additional DNA markers for RFLP.

[0171] The D-SLAM polynucleotides can also be used as an alternative toRFLP, by determining the actual base-by-base DNA sequence of selectedportions of an individual's genome. These sequences can be used toprepare PCR primers for amplifying and isolating such selected DNA,which can then be sequenced. Using this technique, individuals can beidentified because each individual will have a unique set of DNAsequences. Once an unique ID database is established for an individual,positive identification of that individual, living or dead, can be madefrom extremely small tissue samples.

[0172] Forensic biology also benefits from using DNA-basedidentification techniques as disclosed herein. DNA sequences taken fromvery small biological samples such as tissues, e.g., hair or skin, orbody fluids, e.g., blood, saliva, semen, etc., can be amplified usingPCR. In one prior art technique, gene sequences amplified frompolymorphic loci, such as DQa class II HLA gene, are used in forensicbiology to identify individuals. (Erlich, H., PCR Technology, Freemanand Co. (1992).) Once these specific polymorphic loci are amplified,they are digested with one or more restriction enzymes, yielding anidentifying set of bands on a Southern blot probed with DNAcorresponding to the DQa class II HLA gene. Similarly, D-SLAMpolynucleotides can be used as polymorphic markers for forensicpurposes.

[0173] There is also a need for reagents capable of identifying thesource of a particular tissue. Such need arises, for example, inforensics when presented with tissue of unknown origin. Appropriatereagents can comprise, for example, DNA probes or primers specific toparticular tissue prepared from D-SLAM sequences. Panels of suchreagents can identify tissue by species and/or by organ type. In asimilar fashion, these reagents can be used to screen tissue culturesfor contamination.

[0174] Because D-SLAM is found expressed in dendritic cells, T celllymphoma, lymph node, spleen, thymus, small intestine, and uterus,D-SLAM polynucleotides are useful as hybridization probes fordifferential identification of the tissue(s) or cell type(s) present ina biological sample. Similarly, polypeptides and antibodies directed toD-SLAM polypeptides are useful to provide immunological probes fordifferential identification of the tissue(s) or cell type(s). Inaddition, for a number of disorders of the above tissues or cells,particularly of the immune system, significantly higher or lower levelsof D-SLAM gene expression may be detected in certain tissues (e.g.,cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma,urine, synovial fluid or spinal fluid) taken from an individual havingsuch a disorder, relative to a “standard” D-SLAM gene expression level,i.e., the D-SLAM expression level in healthy tissue from an individualnot having the immune system disorder.

[0175] Thus, the invention provides a diagnostic method of a disorder,which involves: (a) assaying D-SLAM gene expression level in cells orbody fluid of an individual; (b) comparing the D-SLAM gene expressionlevel with a standard D-SLAM gene expression level, whereby an increaseor decrease in the assayed D-SLAM gene expression level compared to thestandard expression level is indicative of disorder in the immunesystem.

[0176] In the very least, the D-SLAM polynucleotides can be used asmolecular weight markers on Southern gels, as diagnostic probes for thepresence of a specific mRNA in a particular cell type, as a probe to“subtract-out” known sequences in the process of discovering novelpolynucleotides, for selecting and making oligomers for attachment to a“gene chip” or other to raise anti-DNA antibodies using DNA immunizationtechniques, and as an antigen to immune response.

[0177] Uses D-SLAM Polypeptides

[0178] D-SLAM polypeptides can be used in numerous ways. The followingdescription should be considered exemplary and utilizes knowntechniques.

[0179] D-SLAM polypeptides can be used to assay protein levels in abiological sample using antibody-based techniques. For example, proteinexpression in tissues can be studied with classical immunohistologicalmethods. (Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985);Jalkanen, M., et al., J. Cell . Biol. 105:3087-3096 (1987).) Otherantibody-based methods useful for deducting protein gene expressioninclude immunoassays, such as the enzyme linked immunosorbent assay(ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labelsare known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

[0180] In addition to assaying secreted protein levels in a biologicalsample, proteins can also be detected in vivo by imaging. Antibodylabels or markers for in vivo imaging of protein include thosedetectable by X-radiography, NMR or ESR. For X-radiography, suitablelabels include radioisotopes such as barium or cesium, which emitdetectable radiation but are not overtly harmful to the subject.Suitable markers for NMR and ESR include those with a detectablecharacteristic spin, such as deuterium, which may be incorporated intothe antibody by labeling of nutrients for the relevant hybridoma.

[0181] A protein-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, 131I, 112In, 99mTc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously, orintraperitoneally) into the mammal. It will be understood in the artthat the size of the subject and the imaging system used will determinethe quantity of imaging moiety needed to produce diagnostic images. Inthe case of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of 99mTc. The labeled antibody or antibody fragment willthen preferentially accumulate at the location of cells which containthe specific protein. In vivo tumor imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments.” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982).)

[0182] Thus, the invention provides a diagnostic method of a disorder,which involves (a) assaying the expression of D-SLAM polypeptide incells or body fluid of an individual; (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed D-SLAM polypeptide gene expression levelcompared to the standard expression level is indicative of a disorder.

[0183] Moreover, D-SLAM polypeptides can be used to treat disease. Forexample, patients can be administered D-SLAM polypeptides in an effortto replace absent or decreased levels of the D-SLAM polypeptide (e.g.,insulin), to supplement absent or decreased levels of a differentpolypeptide (e.g., hemoglobin S for hemoglobin B), to inhibit theactivity of a polypeptide (e.g., an oncogene), to activate the activityof a polypeptide (e.g., by binding to a receptor), to reduce theactivity of a membrane bound receptor by competing with it for freeligand (e.g., soluble TNF receptors used in reducing inflammation), orto bring about a desired response (e.g., blood vessel growth).

[0184] Similarly, antibodies directed to D-SLAM polypeptides can also beused to treat disease. For example, administration of an antibodydirected to a D-SLAM polypeptide can bind and reduce overproduction ofthe polypeptide. Similarly, administration of an antibody can activatethe polypeptide, such as by binding to a polypeptide bound to a membrane(receptor).

[0185] At the very least, the D-SLAM polypeptides can be used asmolecular weight markers on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart. D-SLAM polypeptides can also be used to raise antibodies, which inturn are used to measure protein expression from a recombinant cell, asa way of assessing transformation of the host cell. Moreover, D-SLAMpolypeptides can be used to test the following biological activities.

[0186] Gene Therapy Methods

[0187] Another aspect of the present invention is to gene therapymethods for treating disorders, diseases and conditions. The genetherapy methods relate to the introduction of nucleic acid (DNA, RNA andantisense DNA or RNA) sequences into an animal to achieve expression ofthe D-SLAM polypeptide of the present invention. This method requires apolynucleotide which codes for a D-SLAM polypeptide operatively linkedto a promoter and any other genetic elements necessary for theexpression of the polypeptide by the target tissue. Such gene therapyand delivery techniques are known in the art, see, for example,WO90/11092, which is herein incorporated by reference.

[0188] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to aD-SLAM polynucleotide ex vivo, with the engineered cells then beingprovided to a patient to be treated with the polypeptide. Such methodsare well-known in the art. For example, see Belldegrun, A., et al., J.Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., CancerResearch 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995);Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato,L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al.,Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer GeneTherapy 3: 31-38 (1996)), which are herein incorporated by reference. Inone embodiment, the cells which are engineered are arterial cells. Thearterial cells may be reintroduced into the patient through directinjection to the artery, the tissues surrounding the artery, or throughcatheter injection.

[0189] As discussed in more detail below, the D-SLAM polynucleotideconstructs can be delivered by any method that delivers injectablematerials to the cells of an animal, such as, injection into theinterstitial space of tissues (heart, muscle, skin, lung, liver, and thelike). The D-SLAM polynucleotide constructs may be delivered in apharmaceutically acceptable liquid or aqueous carrier.

[0190] In one embodiment, the D-SLAM polynucleotide is delivered as anaked polynucleotide. The term “naked” polynucleotide, DNA or RNA refersto sequences that are free from any delivery vehicle that acts toassist, promote or facilitate entry into the cell, including viralsequences, viral particles, liposome formulations, lipofectin orprecipitating agents and the like. However, the D-SLAM polynucleotidescan also be delivered in liposome formulations and lipofectinformulations and the like can be prepared by methods well known to thoseskilled in the art. Such methods are described, for example, in U.S.Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are hereinincorporated by reference.

[0191] The D-SLAM polynucleotide vector constructs used in the genetherapy method are preferably constructs that will not integrate intothe host genome nor will they contain sequences that allow forreplication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

[0192] Any strong promoter known to those skilled in the art can be usedfor driving the expression of D-SLAM DNA. Suitable promoters includeadenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe mMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs; the b-actin promoter; and human growthhormone promoters. The promoter also may be the native promoter forD-SLAM.

[0193] Unlike other gene therapy techniques, one major advantage ofintroducing naked nucleic acid sequences into target cells is thetransitory nature of the polynucleotide synthesis in the cells. Studieshave shown that non-replicating DNA sequences can be introduced intocells to provide production of the desired polypeptide for periods of upto six months.

[0194] The D-SLAM polynucleotide construct can be delivered to theinterstitial space of tissues within the an animal, including of muscle,skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular, fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

[0195] For the naked acid sequence injection, an effective dosage amountof DNA or RNA will be in the range of from about 0.05 mg/kg body weightto about 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

[0196] The preferred route of administration is by the parenteral routeof injection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked D-SLAM DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

[0197] The naked polynucleotides are delivered by any method known inthe art, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, and so-called “gene guns”. These delivery methods are known inthe art.

[0198] As is evidenced in the Examples, naked D-SLAM nucleic acidsequences can be administered in vivo results in the successfulexpression of D-SLAM polypeptide in the femoral arteries of rabbits.

[0199] The constructs may also be delivered with delivery vehicles suchas viral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

[0200] In certain embodiments, the D-SLAM polynucleotide constructs arecomplexed in a liposome preparation. Liposomal preparations for use inthe instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA(1987) 84:7413-7416, which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which isherein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which is hereinincorporated by reference), in functional form.

[0201] Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated byreference). Other commercially available liposomes include transfectace(DDAB/DOPE) and DOTAP/DOPE (Boehringer).

[0202] Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication No. WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., P. Felgneret al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

[0203] Similarly, anionic and neutral liposomes are readily available,such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easilyprepared using readily available materials. Such materials includephosphatidyl, choline, cholesterol, phosphatidyl ethanolamine,dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol(DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. Thesematerials can also be mixed with the DOTMA and DOTAP starting materialsin appropriate ratios. Methods for making liposomes using thesematerials are well known in the art.

[0204] For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

[0205] The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology (1983), 101:512-527, which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca²⁺-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilsonet al., Cell (1979) 17:77); ether injection (Deamer, D. and Bangham, A.,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); detergent dialysis (Enoch, H. and Strittmatter, P.,Proc. Natl. Acad. Sci. USA (1979) 76:145); and reverse-phase evaporation(REV) (Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka, F. andPapahadjopoulos, D., Proc. Natl. Acad. Sci. USA (1978) 75:145;Schaefer-Ridder et al., Science (1982) 215:166), which are hereinincorporated by reference.

[0206] Generally, the ratio of DNA to liposomes will be from about 10:1to about 1:10. Preferably, the ration will be from about 5:1 to about1:5. More preferably, the ration will be about 3:1 to about 1:3. Stillmore preferably, the ratio will be about 1:1.

[0207] U.S. Pat. No. 5,676,954 (which is herein incorporated byreference) reports on the injection of genetic material, complexed withcationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355,4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication no. WO 94/9469 (which areherein incorporated by reference) provide cationic lipids for use intransfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466,5,693,622, 5,580,859, 5,703,055, and international publication no. WO94/9469 (which are herein incorporated by reference) provide methods fordelivering DNA-cationic lipid complexes to mammals.

[0208] In certain embodiments, cells are be engineered, ex vivo or invivo, using a retroviral particle containing RNA which comprises asequence encoding D-SLAM. Retroviruses from which the retroviral plasmidvectors may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, Rous sarcoma Virus, HarveySarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, humanimmunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammarytumor virus.

[0209] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy 1:5-14 (1990), which is incorporated herein by reference in itsentirety. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

[0210] The producer cell line generates infectious retroviral vectorparticles which include polynucleotide encoding D-SLAM. Such retroviralvector particles then may be employed, to transduce eukaryotic cells,either in vitro or in vivo. The transduced eukaryotic cells will expressD-SLAM.

[0211] In certain other embodiments, cells are engineered, ex vivo or invivo, with D-SLAM polynucleotide contained in an adenovirus vector.Adenovirus can be manipulated such that it encodes and expresses D-SLAM,and at the same time is inactivated in terms of its ability to replicatein a normal lytic viral life cycle. Adenovirus expression is achievedwithout integration of the viral DNA into the host cell chromosome,thereby alleviating concerns about insertional mutagenesis. Furthermore,adenoviruses have been used as live enteric vaccines for many years withan excellent safety profile (Schwartz, A. R. et al. (1974) Am. Rev.Respir. Dis.109:233-238). Finally, adenovirus mediated gene transfer hasbeen demonstrated in a number of instances including transfer ofalpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M.A. et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell68:143-155). Furthermore, extensive studies to attempt to establishadenovirus as a causative agent in human cancer were uniformly negative(Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).

[0212] Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992);Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al.,Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

[0213] Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, for example, the HARP promoter of the present invention, butcannot replicate in most cells. Replication deficient adenoviruses maybe deleted in one or more of all or a portion of the following genes:E1a, E1b, E3, E4, E2a, or L1 through L5.

[0214] In certain other embodiments, the cells are engineered, ex vivoor in vivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol.158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

[0215] For example, an appropriate AAV vector for use in the presentinvention will include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The D-SLAM polynucleotideconstruct is inserted into the AAV vector using standard cloningmethods, such as those found in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAVvector is then transfected into packaging cells which are infected witha helper virus, using any standard technique, including lipofection,electroporation, calcium phosphate precipitation, etc. Appropriatehelper viruses include adenoviruses, cytomegaloviruses, vacciniaviruses, or herpes viruses. Once the packaging cells are transfected andinfected, they will produce infectious AAV viral particles which containthe D-SLAM polynucleotide construct. These viral particles are then usedto transduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the D-SLAM polynucleotide construct integrated intoits genome, and will express D-SLAM.

[0216] Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding D-SLAM) via homologous recombination (see, e.g., U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No.WO 96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989).This method involves the activation of a gene which is present in thetarget cells, but which is not normally expressed in the cells, or isexpressed at a lower level than desired.

[0217] Polynucleotide constructs are made, using standard techniquesknown in the art, which contain the promoter with targeting sequencesflanking the promoter. Suitable promoters are described herein. Thetargeting sequence is sufficiently complementary to an endogenoussequence to permit homologous recombination of the promoter-targetingsequence with the endogenous sequence. The targeting sequence will besufficiently near the 5′ end of the D-SLAM desired endogenouspolynucleotide sequence so the promoter will be operably linked to theendogenous sequence upon homologous recombination.

[0218] The promoter and the targeting sequences can be amplified usingPCR. Preferably, the amplified promoter contains distinct restrictionenzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

[0219] The promoter-targeting sequence construct is delivered to thecells, either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

[0220] The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous D-SLAM sequence is placedunder the control of the promoter. The promoter then drives theexpression of the endogenous D-SLAM sequence.

[0221] The polynucleotides encoding D-SLAM may be administered alongwith other polynucleotides encoding other angiongenic proteins.Angiogenic proteins include, but are not limited to, acidic and basicfibroblast growth factors, VEGF-1, epidermal growth factor alpha andbeta, platelet-derived endothelial cell growth factor, platelet-derivedgrowth factor, tumor necrosis factor alpha, hepatocyte growth factor,insulin like growth factor, colony stimulating factor, macrophage colonystimulating factor, granulocyte/macrophage colony stimulating factor,and nitric oxide synthase.

[0222] Preferably, the polynucleotide encoding D-SLAM contains asecretory signal sequence that facilitates secretion of the protein.Typically, the signal sequence is positioned in the coding region of thepolynucleotide to be expressed towards or at the 5′ end of the codingregion. The signal sequence may be homologous or heterologous to thepolynucleotide of interest and may be homologous or heterologous to thecells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

[0223] Any mode of administration of any of the above-describedpolynucleotides constructs can be used so long as the mode results inthe expression of one or more molecules in an amount sufficient toprovide a therapeutic effect. This includes direct needle injection,systemic injection, catheter infusion, biolistic injectors, particleaccelerators (i.e., “gene guns”), gelfoam sponge depots, othercommercially available depot materials, osmotic pumps (e.g., Alzaminipumps), oral or suppositorial solid (tablet or pill) pharmaceuticalformulations, and decanting or topical applications during surgery. Forexample, direct injection of naked calcium phosphate-precipitatedplasmid into rat liver and rat spleen or a protein-coated plasmid intothe portal vein has resulted in gene expression of the foreign gene inthe rat livers (Kaneda et al., Science 243:375 (1989)).

[0224] A preferred method of local administration is by directinjection. Preferably, a recombinant molecule of the present inventioncomplexed with a delivery vehicle is administered by direct injectioninto or locally within the area of arteries. Administration of acomposition locally within the area of arteries refers to injecting thecomposition centimeters and preferably, millimeters within arteries.

[0225] Another method of local administration is to contact apolynucleotide construct of the present invention in or around asurgical wound. For example, a patient can undergo surgery and thepolynucleotide construct can be coated on the surface of tissue insidethe wound or the construct can be injected into areas of tissue insidethe wound.

[0226] Therapeutic compositions useful in systemic administration,include recombinant molecules of the present invention complexed to atargeted delivery vehicle of the present invention. Suitable deliveryvehicles for use with systemic administration comprise liposomescomprising ligands for targeting the vehicle to a particular site.

[0227] Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

[0228] Determining an effective amount of substance to be delivered candepend upon a number of factors including, for example, the chemicalstructure and biological activity of the substance, the age and weightof the animal, the precise condition requiring treatment and itsseverity, and the route of administration. The frequency of treatmentsdepends upon a number of factors, such as the amount of polynucleotideconstructs administered per dose, as well as the health and history ofthe subject. The precise amount, number of doses, and timing of doseswill be determined by the attending physician or veterinarian.

[0229] Therapeutic compositions of the present invention can beadministered to any animal, preferably to mammals and birds. Preferredmammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle,horses and pigs, with humans being particularly preferred.

[0230] Biological Activities of D-SLAM

[0231] D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, can be used in assays to test for one or morebiological activities. If D-SLAM polynucleotides or polypeptides, oragonists or antagonists of D-SLAM, do exhibit activity in a particularassay, it is likely that D-SLAM may be involved in the diseasesassociated with the biological activity. Therefore, D-SLAM could be usedto treat the associated disease.

[0232] D-SLAM is a cell surface receptor homologous to members of theSecreted Lymphocyte Activation Molecule (SLAM) family, and thus shouldhave activity similar to other SLAM family members. Current studies inthe literature demonstrate that SLAM can associate with itself, and thatthis homotypic interaction can activate B- and T-cells. Therefore,D-SLAM may interact specifically with SLAM, with D-SLAM (a homotypicinteraction), or other B- and T-cell receptor molecules on the surfaceof B- and T-cells to affect the activation, proliferation, survival,and/or differentiation of immune cells. Similarly, soluble D-SLAM may bean important costimulatory molecule for therapeutic uses or immunemodulation. Ligands, such as antibodies, may mimic the action of solubleD-SLAM by binding to D-SLAM, SLAM, or other dendritic cell receptors.

[0233] Binding of D-SLAM induces the production of interferon-gamma fromother cell types, particularly T- and B-cells (data not shown.) Thebinding may occur through homotypic association with membrane boundD-SLAM, association with SLAM, or association with other T- or B-cellreceptors. Ligands, such as antibodies, may mimic the induction ofinterferon-gamma by soluble D-SLAM by binding to D-SLAM, SLAM, or otherdendritic cell receptors.

[0234] Moreover, because of the tissue distribution of D-SLAM, thisprotein may also play a role in stimulating dendritic or antigenpresenting cells. For example, a secreted form of D-SLAM, containing theextracellular domain or the full-length form, may bind to and stimulateD-SLAM molecules located on the surface of dendritic orantigen-presenting cells in homotypic manner. Binding may also occur toSLAM, or other dendritic cell surface receptors. This binding mayregulate the survival, proliferation, differentiation, activation ormaturation of dendritic cells or antigen presenting cells, effectingantigen recognition and immune response. Moreover, ligands, such asantibodies, may mimic the action of soluble D-SLAM by binding to D-SLAM,SLAM, or other dendritic cell receptors.

[0235] Thus, D-SLAM may be useful as a therapeutic molecule. It could beused to control the proliferation, activation, maturation, survival,and/or differentiation of hematopoietic cells, in particular B- andT-cells. Particularly, D-SLAM may be a useful therapeutic to mediateimmune modulation, and may influence the Th0-TH1-TH2 profile of apatient's immune system. For example, D-SLAM may drive immune responseto the Th0-TH 1 pathway. This control of immune cells would beparticularly important in the treatment of immune disorders, such asautoimmune diseases or immunosuppression (see below). Preferably,treatment of immune disorders could be carried out using a secreted formof D-SLAM, gene therapy, or ex vivo applications. Moreover, inhibitorsof D-SLAM, either blocking antibodies or mutant forms, could modulatethe expression of D-SLAM. These inhibitors may be useful to treatdiseases associated with the misregulation of D-SLAM, such as T celllymphoma.

[0236] Immune Activity 1

[0237] D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may be useful in treating deficiencies ordisorders of the immune system, by activating or inhibiting theproliferation, differentiation, or mobilization (chemotaxis) of immunecells. Immune cells develop through a process called hematopoiesis,producing myeloid (platelets, red blood cells, neutrophils, andmacrophages) and lymphoid (B and T lymphocytes) cells from pluripotentstem cells. The etiology of these immune deficiencies or disorders maybe genetic, somatic, such as cancer or some autoimmune disorders,acquired (e.g., by chemotherapy or toxins), or infectious. Moreover,D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, can be used as a marker or detector of a particular immunesystem disease or disorder.

[0238] D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may be useful in treating or detectingdeficiencies or disorders of hematopoietic cells. D-SLAM polynucleotidesor polypeptides, or agonists or antagonists of D-SLAM, could be used toincrease differentiation and proliferation of hematopoietic cells,including the pluripotent stem cells, in an effort to treat thosedisorders associated with a decrease in certain (or many) typeshematopoietic cells. Examples of immunologic deficiency syndromesinclude, but are not limited to: blood protein disorders (e.g.agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, commonvariable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLVinfection, leukocyte adhesion deficiency syndrome, lymphopenia,phagocyte bactericidal dysfunction, severe combined immunodeficiency(SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, orhemoglobinuria.

[0239] Moreover, D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, can also be used to modulate hemostatic (thestopping of bleeding) or thrombolytic activity (clot formation). Forexample, by increasing hemostatic or thrombolytic activity, D-SLAMpolynucleotides or polypeptides, or agonists or antagonists of D-SLAM,could be used to treat blood coagulation disorders (e.g.,afibrinogenemia, factor deficiencies), blood platelet disorders (e.g.thrombocytopenia), or wounds resulting from trauma, surgery, or othercauses. Alternatively, D-SLAM polynucleotides or polypeptides, oragonists or antagonists of D-SLAM, that can decrease hemostatic orthrombolytic activity could be used to inhibit or dissolve clotting,important in the treatment of heart attacks (infarction), strokes, orscarring.

[0240] D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may also be useful in treating or detectingautoimmune disorders. Many autoimmune disorders result frominappropriate recognition of self as foreign material by immune cells.This inappropriate recognition results in an immune response leading tothe destruction of the host tissue. Therefore, the administration ofD-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, that can inhibit an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune disorders.

[0241] Examples of autoimmune disorders that can be treated or detectedinclude, but are not limited to: Addison's Disease, hemolytic anemia,antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergicencephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves'Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia,Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter'sDisease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic LupusErythematosus, Autoimmune Pulmonary Inflammation, Guillain-BarreSyndrome, insulin dependent diabetes mellitis, and autoimmuneinflammatory eye disease.

[0242] Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM. Moreover, these molecules can be used to treatanaphylaxis, hypersensitivity to an antigenic molecule, or blood groupincompatibility.

[0243] D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may also be used to treat and/or prevent organrejection or graft-versus-host disease (GVHD). Organ rejection occurs byhost immune cell destruction of the transplanted tissue through animmune response. Similarly, an immune response is also involved in GVHD,but, in this case, the foreign transplanted immune cells destroy thehost tissues. The administration of D-SLAM polynucleotides orpolypeptides, or agonists or antagonists of D-SLAM, that inhibits animmune response, particularly the proliferation, differentiation, orchemotaxis of T-cells, may be an effective therapy in preventing organrejection or GVHD.

[0244] Similarly, D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may also be used to modulate inflammation. Forexample, D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may inhibit the proliferation and differentiationof cells involved in an inflammatory response. These molecules can beused to treat inflammatory conditions, both chronic and acuteconditions, including inflammation associated with infection (e.g.,septic shock, sepsis, or systemic inflammatory response syndrome(SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL-1.)

[0245] Hyperproliferative Disorders

[0246] D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, can be used to treat or detect hyperproliferativedisorders, including neoplasms. D-SLAM polynucleotides or polypeptides,or agonists or antagonists of D-SLAM, may inhibit the proliferation ofthe disorder through direct or indirect interactions. Alternatively,D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, may proliferate other cells which can inhibit thehyperproliferative disorder.

[0247] For example, by increasing an immune response, particularlyincreasing antigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating hyperproliferative disorders,such as a chemotherapeutic agent.

[0248] Examples of hyperproliferative disorders that can be treated ordetected by D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, include, but are not limited to neoplasms locatedin the: abdomen, bone, breast, digestive system, liver, pancreas,peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous (centraland peripheral), lymphatic system, pelvic, skin, soft tissue, spleen,thoracic, and urogenital.

[0249] Similarly, other hyperproliferative disorders can also be treatedor detected by D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM. Examples of such hyperproliferative disordersinclude, but are not limited to: hypergammaglobulinemia,lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis,Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease,histiocytosis, and any other hyperproliferative disease, besidesneoplasia, located in an organ system listed above.

[0250] Cardiovascular Disorders

[0251] D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, encoding D-SLAM may be used to treatcardiovascular disorders, including peripheral artery disease, such aslimb ischemia.

[0252] Cardiovascular disorders include cardiovascular abnormalities,such as arterio-arterial fistula, arteriovenous fistula, cerebralarteriovenous malformations, congenital heart defects, pulmonaryatresia, and Scimitar Syndrome. Congenital heart defects include aorticcoarctation, cor triatriatum, coronary vessel anomalies, crisscrossheart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly,Eisemnenger complex, hypoplastic left heart syndrome, levocardia,tetralogy of fallot, transposition of great vessels, double outlet rightventricle, tricuspid atresia, persistent truncus arteriosus, and heartseptal defects, such as aortopulmonary septal defect, endocardialcushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricularheart septal defects.

[0253] Cardiovascular disorders also include heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

[0254] Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

[0255] Heart valve disease include aortic valve insufficiency, aorticvalve stenosis, hear murmurs, aortic valve prolapse, mitral valveprolapse, tricuspid valve prolapse, mitral valve insufficiency, mitralvalve stenosis, pulmonary atresia, pulmonary valve insufficiency,pulmonary valve stenosis, tricuspid atresia, tricuspid valveinsufficiency, and tricuspid valve stenosis.

[0256] Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

[0257] Myocardial ischemias include coronary disease, such as anginapectoris, coronary aneurysm, coronary arteriosclerosis, coronarythrombosis, coronary vasospasm, myocardial infarction and myocardialstunning.

[0258] Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

[0259] Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

[0260] Arterial occlusive diseases include arteriosclerosis,intermittent claudication, carotid stenosis, fibromuscular dysplasias,mesenteric vascular occlusion, Moyamoya disease, renal arteryobstruction, retinal artery occlusion, and thromboangiitis obliterans.

[0261] Cerebrovascular disorders include carotid artery diseases,cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebralartery diseases, cerebral embolism and thrombosis, carotid arterythrombosis, sinus thrombosis, Wallenberg's syndrome, cerebralhemorrhage, epidural hematoma, subdural hematoma, subaraxhnoidhemorrhage, cerebral infarction, cerebral ischemia (includingtransient), subclavian steal syndrome, periventricular leukomalacia,vascular headache, cluster headache, migraine, and vertebrobasilarinsufficiency.

[0262] Embolisms include air embolisms, amniotic fluid embolisms,cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonaryembolisms, and thromoboembolisms. Thrombosis include coronarythrombosis, hepatic vein thrombosis, retinal vein occlusion, carotidartery thrombosis, sinus thrombosis, Wallenberg's syndrome, andthrombophlebitis.

[0263] Ischemia includes cerebral ischemia, ischemic colitis,compartment syndromes, anterior compartment syndrome, myocardialischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitisincludes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

[0264] D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, are especially effective for the treatment ofcritical limb ischemia and coronary disease. As shown in the Examples,administration of D-SLAM polynucleotides and polypeptides to anexperimentally induced ischemia rabbit hindlimb may restore bloodpressure ratio, blood flow, angiographic score, and capillary density.

[0265] D-SLAM polypeptides may be administered using any method known inthe art, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, biolistic injectors, particle accelerators, gelfoam spongedepots, other commercially available depot materials, osmotic pumps,oral or suppositorial solid pharmaceutical formulations, decanting ortopical applications during surgery, aerosol delivery. Such methods areknown in the art. D-SLAM polypeptides may be administered as part of apharmaceutical composition, described in more detail below. Methods ofdelivering D-SLAM polynucleotides are described in more detail herein.

[0266] Anti-Angiogenesis Activity

[0267] The naturally occurring balance between endogenous stimulatorsand inhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkmanet al., N. Engl. J Med., 333:1757-1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985);Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science221:719-725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442-447 (1987).

[0268] The present invention provides for treatment of diseases ordisorders associated with neovascularization by administration of theD-SLAM polynucleotides and/or polypeptides of the invention, as well asagonists or antagonists of D-SLAM. Malignant and metastatic conditionswhich can be treated with the polynucleotides and polypeptides, oragonists or antagonists of the invention include, but are not limitedto, malignancies, solid tumors, and cancers described herein andotherwise known in the art (for a review of such disorders, see Fishmanet al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).

[0269] Ocular disorders associated with neovascularization which can betreated with the D-SLAM polynucleotides and polypeptides of the presentinvention (including D-SLAM agonists and/or antagonists) include, butare not limited to: neovascular glaucoma, diabetic retinopathy,retinoblastoma, retrolental fibroplasia, uveitis, retinopathy ofprematurity macular degeneration, corneal graft neovascularization, aswell as other eye inflammatory diseases, ocular tumors and diseasesassociated with choroidal or iris neovascularization. See, e.g., reviewsby Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al.,Surv. Ophthal. 22:291-312 (1978).

[0270] Additionally, disorders which can be treated with the D-SLAMpolynucleotides and polypeptides of the present invention (includingD-SLAM agonist and/or antagonists) include, but are not limited to,hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques,delayed wound healing, granulations, hemophilic joints, hypertrophicscars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma,scleroderma, trachoma, and vascular adhesions.

[0271] Moreover, disorders and/or states, which can be treated with betreated with the D-SLAM polynucleotides and polypeptides of the presentinvention (including D-SLAM agonist and/or antagonists) include, but arenot limited to, solid tumors, blood born tumors such as leukemias, tumormetastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas,acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas,rheumatoid arthritis, psoriasis, ocular angiogenic diseases, forexample, diabetic retinopathy, retinopathy of prematurity, maculardegeneration, corneal graft rejection, neovascular glaucoma, retrolentalfibroplasia, rubeosis, retinoblastoma, and uvietis, delayed woundhealing, endometriosis, vascluogenesis, granulations, hypertrophic scars(keloids), nonunion fractures, scleroderma, trachoma, vascularadhesions, myocardial angiogenesis, coronary collaterals, cerebralcollaterals, arteriovenous malformations, ischemic limb angiogenesis,Osler-Webber Syndrome, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma fibromuscular dysplasia, woundgranulation, Crohn's disease, atherosclerosis, birth control agent bypreventing vascularization required for embryo implantation controllingmenstruation, diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa),ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

[0272] Diseases at the Cellular Level

[0273] Diseases associated with increased cell survival or theinhibition of apoptosis that could be treated or detected by D-SLAMpolynucleotides or polypeptides, as well as antagonists or agonists ofD-SLAM, include cancers (such as follicular lymphomas, carcinomas withp53 mutations, and hormone-dependent tumors, including, but not limitedto colon cancer, cardiac tumors, pancreatic cancer, melanoma,retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicularcancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma,endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), inflammation, graft v. host disease, acute graftrejection, and chronic graft rejection. In preferred embodiments, D-SLAMpolynucleotides, polypeptides, and/or antagonists of the invention areused to inhibit growth, progression, and/or metasis of cancers, inparticular those listed above.

[0274] Additional diseases or conditions associated with increased cellsurvival that could be treated or detected by D-SLAM polynucleotides orpolypeptides, or agonists or antagonists of D-SLAM, include, but are notlimited to, progression, and/or metastases of malignancies and relateddisorders such as leukemia (including acute leukemias (e.g., acutelymphocytic leukemia, acute myelocytic leukemia (including myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) andchronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia andchronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumorsincluding, but not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

[0275] Diseases associated with increased apoptosis that could betreated or detected by D-SLAM polynucleotides or polypeptides, as wellas agonists or antagonists of D-SLAM, include AIDS; neurodegenerativedisorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration andbrain tumor or prior associated disease); autoimmune disorders (such as,multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) myclodysplastic syndromes (such as aplastic anemia), graft v.host disease, ischemic injury (such as that caused by myocardialinfarction, stroke and reperfusion injury), liver injury (e.g.,hepatitis related liver injury, ischemia/reperfusion injury, cholestosis(bile duct injury) and liver cancer); toxin-induced liver disease (suchas that caused by alcohol), septic shock, cachexia and anorexia.

[0276] Wound Healing and Epithelial Cell Proliferation

[0277] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing D-SLAM polynucleotides orpolypeptides, as well as agonists or antagonists of D-SLAM, fortherapeutic purposes, for example, to stimulate epithelial cellproliferation and basal keratinocytes for the purpose of wound healing,and to stimulate hair follicle production and healing of dermal wounds.D-SLAM polynucleotides or polypeptides, as well as agonists orantagonists of D-SLAM, may be clinically useful in stimulating woundhealing including surgical wounds, excisional wounds, deep woundsinvolving damage of the dermis and epidermis, eye tissue wounds, dentaltissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers,cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resultingfrom heat exposure or chemicals, and other abnormal wound healingconditions such as uremia, malnutrition, vitamin deficiencies andcomplications associted with systemic treatment with steroids, radiationtherapy and antineoplastic drugs and antimetabolites. D-SLAMpolynucleotides or polypeptides, as well as agonists or antagonists ofD-SLAM, could be used to promote dermal reestablishment subsequent todermal loss.

[0278] D-SLAM polynucleotides or polypeptides, as well as agonists orantagonists of D-SLAM, could be used to increase the adherence of skingrafts to a wound bed and to stimulate re-epithelialization from thewound bed. The following are types of grafts that D-SLAM polynucleotidesor polypeptides, agonists or antagonists of D-SLAM, could be used toincrease adherence to a wound bed: autografts, artificial skin,allografts, autodermic graft, autoepdermic grafts, avacular grafts,Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft,delayed graft, dermic graft, epidermic graft, fascia graft, fullthickness graft, heterologous graft, xenograft, homologous graft,hyperplastic graft, lamellar graft, mesh graft, mucosal graft,Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft,penetrating graft, split skin graft, thick split graft. D-SLAMpolynucleotides or polypeptides, as well as agonists or antagonists ofD-SLAM, can be used to promote skin strength and to improve theappearance of aged skin.

[0279] It is believed that D-SLAM polynucleotides or polypeptides, aswell as agonists or antagonists of D-SLAM, will also produce changes inhepatocyte proliferation, and epithelial cell proliferation in the lung,breast, pancreas, stomach, small intesting, and large intestine. D-SLAMpolynucleotides or polypeptides, as well as agonists or antagonists ofD-SLAM, could promote proliferation of epithelial cells such assebocytes, hair follicles, hepatocytes, type II pneumocytes,mucin-producing goblet cells, and other epithelial cells and theirprogenitors contained within the skin, lung, liver, and gastrointestinaltract. D-SLAM polynucleotides or polypeptides, agonists or antagonistsof D-SLAM, may promote proliferation of endothelial cells,keratinocytes, and basal keratinocytes.

[0280] D-SLAM polynucleotides or polypeptides, as well as agonists orantagonists of D-SLAM, could also be used to reduce the side effects ofgut toxicity that result from radiation, chemotherapy treatments orviral infections. D-SLAM polynucleotides or polypeptides, as well asagonists or antagonists of D-SLAM, may have a cytoprotective effect onthe small intestine mucosa. D-SLAM polynucleotides or polypeptides, aswell as agonists or antagonists of D-SLAM, may also stimulate healing ofmucositis (mouth ulcers) that result from chemotherapy and viralinfections.

[0281] D-SLAM polynucleotides or polypeptides, as well as agonists orantagonists of D-SLAM, could further be used in full regeneration ofskin in full and partial thickness skin defects, including bums, (i.e.,repopulation of hair follicles, sweat glands, and sebaceous glands),treatment of other skin defects such as psoriasis. D-SLAMpolynucleotides or polypeptides, as well as agonists or antagonists ofD-SLAM, could be used to treat epidermolysis bullosa, a defect inadherence of the epidermis to the underlying dermis which results infrequent, open and painful blisters by accelerating reepithelializationof these lesions. D-SLAM polynucleotides or polypeptides, as well asagonists or antagonists of D-SLAM, could also be used to treat gastricand doudenal ulcers and help heal by scar formation of the mucosallining and regeneration of glandular mucosa and duodenal mucosal liningmore rapidly. Inflamamatory bowel diseases, such as Crohn's disease andulcerative colitis, are diseases which result in destruction of themucosal surface of the small or large intestine, respectively. Thus,D-SLAM polynucleotides or polypeptides, as well as agonists orantagonists of D-SLAM, could be used to promote the resurfacing of themucosal surface to aid more rapid healing and to prevent progression ofinflammatory bowel disease. Treatment with D-SLAM polynucleotides orpolypeptides, agonists or antagonists of D-SLAM, is expected to have asignificant effect on the production of mucus throughout thegastrointestinal tract and could be used to protect the intestinalmucosa from injurious substances that are ingested or following surgery.D-SLAM polynucleotides or polypeptides, as well as agonists orantagonists of D-SLAM, could be used to treat diseases associate withthe under expression of D-SLAM.

[0282] Moreover, D-SLAM polynucleotides or polypeptides, as well asagonists or antagonists of D-SLAM, could be used to prevent and healdamage to the lungs due to various pathological states. A growth factorsuch as D-SLAM polynucleotides or polypeptides, as well as agonists orantagonists of D-SLAM, which could stimulate proliferation anddifferentiation and promote the repair of alveoli and brochiolarepithelium to prevent or treat acute or chronic lung damage. Forexample, emphysema, which results in the progressive loss of aveoli, andinhalation injuries, i.e., resulting from smoke inhalation and bums,that cause necrosis of the bronchiolar epithelium and alveoli could beeffectively treated using D-SLAM polynucleotides or polypeptides,agonists or antagonists of D-SLAM. Also, D-SLAM polynucleotides orpolypeptides, as well as agonists or antagonists of D-SLAM, could beused to stimulate the proliferation of and differentiation of type IIpneumocytes, which may help treat or prevent disease such as hyalinemembrane diseases, such as infant respiratory distress syndrome andbronchopulmonary displasia, in premature infants.

[0283] D-SLAM polynucleotides or polypeptides, as well as agonists orantagonists of D-SLAM, could stimulate the proliferation anddifferentiation of hepatocytes and, thus, could be used to alleviate ortreat liver diseases and pathologies such as fulminant liver failurecaused by cirrhosis, liver damage caused by viral hepatitis and toxicsubstances (i.e., acetaminophen, carbon tetraholoride and otherhepatotoxins known in the art).

[0284] In addition, D-SLAM polynucleotides or polypeptides, as well asagonists or antagonists of D-SLAM, could be used treat or prevent theonset of diabetes mellitus. In patients with newly diagnosed Types I andII diabetes, where some islet cell function remains, D-SLAMpolynucleotides or polypeptides, as well as agonists or antagonists ofD-SLAM, could be used to maintain the islet function so as to alleviate,delay or prevent permanent manifestation of the disease. Also, D-SLAMpolynucleotides or polypeptides, as well as agonists or antagonists ofD-SLAM, could be used as an auxiliary in islet cell transplantation toimprove or promote islet cell function.

[0285] Infectious Disease

[0286] D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, can be used to treat or detect infectious agents.For example, by increasing the immune response, particularly increasingthe proliferation and differentiation of B and/or T cells, infectiousdiseases may be treated. The immune response may be increased by eitherenhancing an existing immune response, or by initiating a new immuneresponse. Alternatively, D-SLAM polynucleotides or polypeptides, oragonists or antagonists of D-SLAM, may also directly inhibit theinfectious agent, without necessarily eliciting an immune response.

[0287] Viruses are one example of an infectious agent that can causedisease or symptoms that can be treated or detected by D-SLAMpolynucleotides or polypeptides, or agonists or antagonists of D-SLAM.Examples of viruses, include, but are not limited to the following DNAand RNA viral families: Arbovirus, Adenoviridae, Arenaviridae,Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae,Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae(such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus(e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae(e.g., Influenza), Papovaviridae, Parvoviridae, Picornaviridae,Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus),Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g.,Rubivirus). Viruses falling within these families can cause a variety ofdiseases or symptoms, including, but not limited to: arthritis,bronchiollitis, encephalitis, eye infections (e.g., conjunctivitis,keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, ChronicActive, Delta), meningitis, opportunistic infections (e.g., AIDS),pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles,Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia. D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, can be used to treat or detect any of thesesymptoms or diseases.

[0288] Similarly, bacterial or fungal agents that can cause disease orsymptoms and that can be treated or detected by D-SLAM polynucleotidesor polypeptides, or agonists or antagonists of D-SLAM, include, but notlimited to, the following Gram-Negative and Gram-positive bacterialfamilies and fungi: Actinomycetales (e.g., Corynebacterium,Mycobacterium, Norcardia), Aspergillosis, Bacillaceae (e.g., Anthrax,Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia,Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis,Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsiella,Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter,Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae(e.g., Acinetobacter, Gonorrhea, Menigococcal), PasteurellaceaInfections (e.g., Actinobacillus, Heamophilus, Pasteurella),Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, andStaphylococcal. These bacterial or fungal families can cause thefollowing diseases or symptoms, including, but not limited to:bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis,uveitis), gingivitis, opportunistic infections (e.g., AIDS relatedinfections), paronychia, prosthesis-related infections, Reiter'sDisease, respiratory tract infections, such as Whooping Cough orEmpyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery,Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea,meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dernatocycoses), toxemia, urinary tract infections,wound infections. D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, can be used to treat or detect any of thesesymptoms or diseases.

[0289] Moreover, parasitic agents causing disease or symptoms that canbe treated or detected by D-SLAM polynucleotides or polypeptides, oragonists or antagonists of D-SLAM, include, but not limited to, thefollowing families: Amebiasis, Babesiosis, Coccidiosis,Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis,Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas. These parasites can cause a variety ofdiseases or symptoms, including, but not limited to: Scabies,Trombiculiasis, eye infections, intestinal disease (e.g., dysentery,giardiasis), liver disease, lung disease, opportunistic infections(e.g., AIDS related), Malaria, pregnancy complications, andtoxoplasmosis. D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, can be used to treat or detect any of thesesymptoms or diseases.

[0290] Preferably, treatment using D-SLAM polynucleotides orpolypeptides, or agonists or antagonists of D-SLAM, could either be byadministering an effective amount of D-SLAM polypeptide to the patient,or by removing cells from the patient, supplying the cells with D-SLAMpolynucleotide, and returning the engineered cells to the patient (exvivo therapy). Moreover, the D-SLAM polypeptide or polynucleotide can beused as an antigen in a vaccine to raise an immune response againstinfectious disease.

[0291] Regeneration

[0292] D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, can be used to differentiate, proliferate, andattract cells, leading to the regeneration of tissues. (See, Science276:59-87 (1997).) The regeneration of tissues could be used to repair,replace, or protect tissue damaged by congenital defects, trauma(wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis,osteocarthritis, periodontal disease, liver failure), surgery, includingcosmetic plastic surgery, fibrosis, reperfusion injury, or systemiccytokine damage.

[0293] Tissues that could be regenerated using the present inventioninclude organs (e.g., pancreas, liver, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac), vasculature(including vascular and lymphatics), nervous, hematopoietic, andskeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,regeneration occurs without or decreased scarring. Regeneration also mayinclude angiogenesis.

[0294] Moreover, D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may increase regeneration of tissues difficult toheal. For example, increased tendon/ligament regeneration would quickenrecovery time after damage. D-SLAM polynucleotides or polypeptides, oragonists or antagonists of D-SLAM, of the present invention could alsobe used prophylactically in an effort to avoid damage. Specific diseasesthat could be treated include of tendinitis, carpal tunnel syndrome, andother tendon or ligament defects. A further example of tissueregeneration of non-healing wounds includes pressure ulcers, ulcersassociated with vascular insufficiency, surgical, and traumatic wounds.

[0295] Similarly, nerve and brain tissue could also be regenerated byusing D-SLAM polynucleotides or polypeptides, or agonists or antagonistsof D-SLAM, to proliferate and differentiate nerve cells. Diseases thatcould be treated using this method include central and peripheralnervous system diseases, neuropathies, or mechanical and traumaticdisorders (e.g., spinal cord disorders, head trauma, cerebrovasculardisease, and stoke). Specifically, diseases associated with peripheralnerve injuries, peripheral neuropathy (e.g., resulting from chemotherapyor other medical therapies), localized neuropathies, and central nervoussystem diseases (e.g., Alzheimer's disease, Parkinson's disease,Huntington's disease, amyotrophic lateral sclerosis, and Shy-Dragersyndrome), could all be treated using the D-SLAM polynucleotides orpolypeptides, or agonists or antagonists of D-SLAM.

[0296] Chemotaxis

[0297] D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may have chemotaxis activity. A chemotaxicmolecule attracts or mobilizes cells (e.g., monocytes, fibroblasts,neutrophils, T-cells, mast cells, eosinophils, epithelial and/orendothelial cells) to a particular site in the body, such asinflammation, infection, or site of hyperproliferation. The mobilizedcells can then fight off and/or heal the particular trauma orabnormality.

[0298] D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may increase chemotaxic activity of particularcells. These chemotactic molecules can then be used to treatinflammation, infection, hyperproliferative disorders, or any immunesystem disorder by increasing the number of cells targeted to aparticular location in the body. For example, chemotaxic molecules canbe used to treat wounds and other trauma to tissues by attracting immunecells to the injured location. As a chemotactic molecule, D-SLAM couldalso attract fibroblasts, which can be used to treat wounds.

[0299] It is also contemplated that D-SLAM polynucleotides orpolypeptides, or agonists or antagonists of D-SLAM, may inhibitchemotactic activity. These molecules could also be used to treatdisorders. Thus, D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, could be used as an inhibitor of chemotaxis.

[0300] Binding Activity

[0301] D-SLAM polypeptides may be used to screen for molecules that bindto D-SLAM or for molecules to which D-SLAM binds. The binding of D-SLAMand the molecule may activate (agonist), increase, inhibit (antagonist),or decrease activity of the D-SLAM or the molecule bound. Examples ofsuch molecules include antibodies, oligonucleotides, proteins (e.g.,receptors),or small molecules.

[0302] Preferably, the molecule is closely related to the natural ligandof D-SLAM, e.g., a fragment of the ligand, or a natural substrate, aligand, a structural or functional mimetic. (See, Coligan et al.,Current Protocols in Immunology 1(2):Chapter 5 (1991).) Similarly, themolecule can be closely related to the natural receptor to which D-SLAMbinds, or at least, a fragment of the receptor capable of being bound byD-SLAM (e.g., active site). In either case, the molecule can berationally designed using known techniques.

[0303] Preferably, the screening for these molecules involves producingappropriate cells which express D-SLAM, either as a secreted protein oron the cell membrane. Preferred cells include cells from mammals, yeast,Drosophila, or E. coli. Cells expressing D-SLAM (or cell membranecontaining the expressed polypeptide) are then preferably contacted witha test compound potentially containing the molecule to observe binding,stimulation, or inhibition of activity of either D-SLAM or the molecule.

[0304] The assay may simply test binding of a candidate compoundtoD-SLAM, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to D-SLAM.

[0305] Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining D-SLAM, measuring D-SLAM/molecule activity or binding, andcomparing the D-SLAM/molecule activity or binding to a standard.

[0306] Preferably, an ELISA assay can measure D-SLAM level or activityin a sample (e.g., biological sample) using a monoclonal or polyclonalantibody. The antibody can measure D-SLAM level or activity by eitherbinding, directly or indirectly, to D-SLAM or by competing with D-SLAMfor a substrate.

[0307] Additionally, the receptor to which D-SLAM binds can beidentified by numerous methods known to those of skill in the art, forexample, ligand panning and FACS sorting (Coligan, et al., CurrentProtocols in Immun., 1(2), Chapter 5, (1991)). For example, expressioncloning is employed wherein polyadenylated RNA is prepared from a cellresponsive to the polypeptides, for example, NIH3T3 cells which areknown to contain multiple receptors for the FGF family proteins, andSC-3 cells, and a cDNA library created from this RNA is divided intopools and used to transfect COS cells or other cells that are notresponsive to the polypeptides. Transfected cells which are grown onglass slides are exposed to the polypeptide of the present invention,after they have been labelled. The polypeptides can be labeled by avariety of means including iodination or inclusion of a recognition sitefor a site-specific protein kinase.

[0308] Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

[0309] As an alternative approach for receptor identification, thelabeled polypeptides can be photoaffinity linked with cell membrane orextract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE analysis and exposed to X-ray film. Thelabeled complex containing the receptors of the polypeptides can beexcised, resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

[0310] Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of D-SLAM therebyeffectively generating agonists and antagonists of D-SLAM. Seegenerally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252,and 5,837,458, and Patten, P. A., et al, Curr. Opinion Biotechnol.8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998);Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M.M. and Blasco, R. Biotechniques 24(2):308-13 (1998) (each of thesepatents and publications are hereby incorporated by reference). In oneembodiment, alteration of D-SLAM polynucleotides and correspondingpolypeptides may be achieved by DNA shuffling. DNA shuffling involvesthe assembly of two or more DNA segments into a desired D-SLAM moleculeby homologous, or site-specific, recombination. In another embodiment,D-SLAM polynucleotides and corresponding polypeptides may be alterred bybeing subjected to random mutagenesis by error-prone PCR, randomnucleotide insertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragrnents, etc., of D-SLAM may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules. In preferred embodiments, the heterologousmolecules are Secreted Lymphocyte Activation Molecule (SLAM) familymembers. In further preferred embodiments, the heterologous molecule isa growth factor such as, for example, platelet-derived growth factor(PDGF), insulin-like growth factor (IGF-I), transforming growth factor(TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor(FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5,BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2,dorsalin, growth differentiation factors (GDFs), nodal, MIS,inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

[0311] Other preferred fragments are biologically active D-SLAMfragments. Biologically active fragments are those exhibiting activitysimilar, but not necessarily identical, to an activity of the D-SLAMpolypeptide. The biological activity of the fragments may include animproved desired activity, or a decreased undesirable activity.

[0312] Additionally, this invention provides a method of screeningcompounds to identify those which modulate the action of the polypeptideof the present invention. An example of such an assay comprisescombining a mammalian fibroblast cell, a the polypeptide of the presentinvention, the compound to be screened and ³[H] thymidine under cellculture conditions where the fibroblast cell would normally proliferate.A control assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of ³[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of ³[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

[0313] In another method, a mammalian cell or membrane preparationexpressing a receptor for a polypeptide of the present invention isincubated with a labeled polypeptide of the present invention in thepresence of the compound. The ability of the compound to enhance orblock this interaction could then be measured. Alternatively, theresponse of a known second messenger system following interaction of acompound to be screened and the D-SLAM receptor is measured and theability of the compound to bind to the receptor and elicit a secondmessenger response is measured to determine if the compound is apotential agonist or antagonist. Such second messenger systems includebut are not limited to, cAMP guanylate cyclase, ion channels orphosphoinositide hydrolysis.

[0314] All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or.to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting the D-SLAM/molecule.Moreover, the assays can discover agents which may inhibit or enhancethe production of D-SLAM from suitably manipulated cells or tissues.Therefore, the invention includes a method of identifying compoundswhich bind to D-SLAM comprising the steps of: (a) incubating a candidatebinding compound with D-SLAM; and (b) determining if binding hasoccurred. Moreover, the invention includes a method of identifyingagonists/antagonists comprising the steps of: (a) incubating a candidatecompound with D-SLAM, (b) assaying a biological activity, and (b)determining if a biological activity of D-SLAM has been altered.

[0315] Also, one could identify molecules bind D-SLAM experimentally byusing the beta-pleated sheet regions disclosed in FIG. 3 and Table 1.Accordingly, specific embodiments of the invention are directed topolynucleotides encoding polypeptides which comprise, or alternativelyconsist of, the amino acid sequence of each beta pleated sheet regionsdisclosed in FIG. 3/Table 1. Additional embodiments of the invention aredirected to polynucleotides encoding D-SLAM polypeptides which comprise,or alternatively consist of, any combination or all of the beta pleatedsheet regions disclosed in FIG. 3/Table 1. Additional preferredembodiments of the invention are directed to polypeptides whichcomprise, or alternatively consist of, the D-SLAM amino acid sequence ofeach of the beta pleated sheet regions disclosed in FIG. 3/Table 1.Additional embodiments of the invention are directed to D-SLAMpolypeptides which comprise, or alternatively consist of, anycombination or all of the beta pleated sheet regions disclosed in FIG.3/Table 1.

[0316] Antisense and Ribozyme (Antagonists)

[0317] In specific embodiments, antagonists according to the presentinvention are nucleic acids corresponding to the sequences contained inSEQ ID NO:1, or the complementary strand thereof, and/or to nucleotidesequences contained in the deposited clone 209623. In one embodiment,antisense sequence is generated internally by the organism, in anotherembodiment, the antisense sequence is separately administered (see, forexample, O'Connor, J., Neurochem. 56:560 (1991). Oligodeoxynucleotidesas Anitsense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Antisense technology can be used to control gene expressionthrough antisense DNA or RNA, or through triple-helix formation.Antisense techniques are discussed for example, in Okano, J., Neurochem.56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

[0318] For example, the 5′ coding portion of a polynucleotide thatencodes the mature polypeptide of the present invention may be used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

[0319] In one embodiment, the D-SLAM antisense nucleic acid of theinvention is produced intracellularly by transcription from an exogenoussequence. For example, a vector or a portion thereof, is transcribed,producing an antisense nucleic acid (RNA) of the invention. Such avector would contain a sequence encoding the D-SLAM antisense nucleicacid. Such a vector can remain episomal or become chromosomallyintegrated, as long as it can be transcribed to produce the desiredantisense RNA. Such vectors can be constructed by recombinant DNAtechnology methods standard in the art. Vectors can be plasmid, viral,or others know in the art, used for replication and expression invertebrate cells. Expression of the sequence encoding D-SLAM, orfragments thereof, can be by any promoter known in the art to act invertebrate, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include, but are not limited to, the SV40early promoter region (Bemoist and Chambon, Nature 29:304-310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell 22:787-797 (1980), the herpes thymidinepromoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445(1981), the regulatory sequences of the metallothionein gene (Brinster,et al., Nature 296:39-42 (1982)), etc.

[0320] The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a D-SLAMgene. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA,”referred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded D-SLAM antisense nucleic acids, a single strandof the duplex DNA may thus be tested, or triplex formation may beassayed. The ability to hybridize will depend on both the degree ofcomplementarity and the length of the antisense nucleic acid. Generally,the larger the hybridizing nucleic acid, the more base mismatches with aD-SLAM RNA it may contain and still form a stable duplex (or triplex asthe case may be). One skilled in the art can ascertain a tolerabledegree of mismatch by use of standard procedures to determine themelting point of the hybridized complex.

[0321] Oligonucleotides that are complementary to the 5′ end of themessage, e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333-335. Thus, oligonucleotides complementary to either the 5′- or3′-non-translated, non-coding regions of D-SLAM shown in FIGS. 1A-Bcould be used in an antisense approach to inhibit translation ofendogenous D-SLAM mRNA. Oligonucleotides complementary to the 5′untranslated region of the mRNA should include the complement of the AUGstart codon. Antisense oligonucleotides complementary to mRNA codingregions are less efficient inhibitors of translation but could be usedin accordance with the invention. Whether designed to hybridize to the5′-, 3′- or coding region of D-SLAM mRNA, antisense nucleic acids shouldbe at least six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Inspecific aspects the oligonucleotide is at least 10 nucleotides, atleast 17 nucleotides, at least 25 nucleotides or at least 50nucleotides.

[0322] The polynucleotides of the invention can be DNA or RNA orchimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone, for example, toimprove stability of the molecule, hybridization, etc. Theoligonucleotide may include other appended groups such as peptides(e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al.,1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO88/09810,published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCTPublication No. WO89/10134, published Apr. 25, 1988),hybridization-triggered cleavage agents. (See, e.g., Krol et al., 1988,BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988,Pharm. Res. 5:539-549). To this end, the oligonucleotide may beconjugated to another molecule, e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, hybridization-triggered cleavageagent, etc.

[0323] The antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including, but not limitedto, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil,. 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0324] The antisense oligonucleotide may also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0325] In yet another embodiment, the antisense oligonucleotidecomprises at least one modified phosphate backbone selected from thegroup including, but not limited to, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[0326] In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

[0327] Polynucleotides of the invention may be synthesized by standardmethods known in the art e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonuceotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

[0328] While antisense nucleotides complementary to the D-SLAM codingregion sequence used, those complementary to the transcribeduntranslated region are most preferred.

[0329] Potential antagonists according to the invention also includecatalytic RNA, or a ribozyme (See, e.g., PCT International PublicationWO 90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy D-SLAM mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that forn complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA have thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, Nature 334:585-591 (1988).There are numerous potential hammerhead ribozyme cleavage sites withinthe nucleotide sequence of D-SLAM (FIGS. 1A-B). Preferably, the ribozymeis engineered so that the cleavage recognition site is located near the5′ end of the D-SLAM mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-fuctional mRNA transcripts.

[0330] As in the antisense approach, the ribozymes of the invention canbe composed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express D-SLAMin vivo. DNA constructs encoding the ribozyme may be introduced into thecell in the same manner as described above for the introduction ofantisense encoding DNA. A preferred method of delivery involves using aDNA construct “encoding” the ribozyme under the control of a strongconstitutive promoter, such as, for example, pol III or pol II promoter,so that transfected cells will produce sufficient quantities of theribozyme to destroy endogenous D-SLAM messages and inhibit translation.Since ribozymes unlike antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

[0331] Antagonist/agonist compounds may be employed to inhibit the cellgrowth and proliferation effects of the polypeptides of the presentinvention on neoplastic cells and tissues, i.e. stimulation ofangiogenesis of tumors, and, therefore, retard or prevent abnormalcellular growth and proliferation, for example, in tumor formation orgrowth.

[0332] The antagonist/agonist may also be employed to preventhyper-vascular diseases, and prevent the proliferation of epitheliallens cells after extracapsular cataract surgery. Prevention of themitogenic activity of the polypeptides of the present invention may alsobe desirous in cases such as restenosis after balloon angioplasty.

[0333] The antagonist/agonist may also be employed to prevent the growthof scar tissue during wound healing.

[0334] The antagonist/agonist may also be employed to treat the diseasesdescribed herein.

[0335] Other Activities

[0336] The polypeptide of the present invention, as a result of theability to stimulate vascular endothelial cell growth, may be employedin treatment for stimulating re-vascularization of ischemic tissues dueto various disease conditions such as thrombosis, arteriosclerosis, andother cardiovascular conditions. These polypeptide may also be employedto stimulate angiogenesis and limb regeneration, as discussed above.

[0337] The polypeptide may also be employed for treating wounds due toinjuries, burns, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

[0338] The polypeptide of the present invention may also be employedstimulate neuronal growth and to treat and prevent neuronal damage whichoccurs in certain neuronal disorders or neuro-degenerative conditionssuch as Alzheimer's disease, Parkinson's disease, and AIDS-relatedcomplex. D-SLAM may have the ability to stimulate chondrocyte growth,therefore, they may be employed to enhance bone and periodontalregeneration and aid in tissue transplants or bone grafts.

[0339] The polypeptide of the present invention may be also be employedto prevent skin aging due to sunburn by stimulating keratinocyte growth.

[0340] The D-SLAM polypeptide may also be employed for preventing hairloss, since FGF family members activate hair-forming cells and promotesmelanocyte growth. Along the same lines, the polypeptides of the presentinvention may be employed to stimulate growth and differentiation ofhematopoietic cells and bone marrow cells when used in combination withother cytokines.

[0341] The D-SLAM polypeptide may also be employed to maintain organsbefore transplantation or for supporting cell culture of primarytissues.

[0342] The polypeptide of the present invention may also be employed forinducing tissue of meodermal origin to differentiate in early embryos.

[0343] D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may also increase or decrease the differentiationor proliferation of embryonic stem cells, besides, as discussed above,hematopoietic lineage.

[0344] D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may also be used to modulate mammaliancharacteristics, such as body height, weight, hair color, eye color,skin, percentage of adipose tissue, pigmentation, size, and shape (e.g.,cosmetic surgery). Similarly, D-SLAM polynucleotides or polypeptides, oragonists or antagonists of D-SLAM, may be used to modulate mammalianmetabolism affecting catabolism, anabolism, processing, utilization, andstorage of energy.

[0345] D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may be used to change a mammal's mental state orphysical state by influencing biorhythms, cardiac rhythms, depression(including depressive disorders), tendency for violence, tolerance forpain, reproductive capabilities (preferably by Activin or Inhibin-likeactivity), hormonal or endocrine levels, appetite, libido, memory,stress, or other cognitive qualities.

[0346] D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may also be used as a food additive orpreservative, such as to increase or decrease storage capabilities, fatcontent, lipid, protein, carbohydrate, vitamins, minerals, cofactors orother nutritional components.

[0347] The above-recited applications have uses in a wide variety ofhosts. Such hosts include but are not limited to, human, murine, rabbit,goat, guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig,chicken, goat, cow, sheep, dog, cat, non-human primate, and human. Inspecific embodiments, the host is a mouse, rabbit, goat, guinea pig,chicken, rat, hamster, pig, sheep, dog or cat. In preferred embodiments,the host is a mammal. In most preferred embodiments, the host is ahuman.

[0348] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

[0349] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLES Example 1 Isolation of the D-SLAM cDNA Clone from the DepositedSample

[0350] The cDNA for D-SLAM is inserted into the SalI/NotI multiplecloning site of pCMVSport 3.0. (Life Technologies, Inc., P. O. Box 6009,Gaithersburg, Md. 20897.) pCMVSport 3.0 contains an ampicillinresistance gene and may be transformed into E. coli strain DH10B, alsoavailable from Life Technologies. (See, for instance, Gruber, C. E., etal., Focus 15:59- (1993).).

[0351] Two approaches can be used to isolate D-SLAM from the depositedsample. First, a specific polynucleotide of SEQ ID NO:1 with 30-40nucleotides is synthesized using an Applied Biosystems DNA synthesizeraccording to the sequence reported. The oligonucleotide is labeled, forinstance, with ³²P-γ-ATP using T4 polynucleotide kinase and purifiedaccording to routine methods. (E.g., Maniatis et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y.(1982).) The plasmid mixture is transformed into a suitable host (suchas XL-1 Blue (Stratagene)) using techniques known to those of skill inthe art, such as those provided by the vector supplier or in relatedpublications or patents. The transformants are plated on 1.5% agarplates (containing the appropriate selection agent, e.g., ampicillin) toa density of about 150 transformants (colonies) per plate. These platesare screened using Nylon membranes according to routine methods forbacterial colony screening (e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Edit., (1989), Cold Spring Harbor LaboratoryPress, pages 1.93 to 1.104), or other techniques known to those of skillin the art.

[0352] Alternatively, two primers of 17-20 nucleotides derived from bothends of the SEQ ID NO:1 (i.e., within the region of SEQ ID NO:1 boundedby the 5′ NT and the 3′ NT of the clone) are synthesized and used toamplify the D-SLAM cDNA using the deposited cDNA plasmid as a template.The polymerase chain reaction is carried out under routine conditions,for instance, in 25 μl of reaction mixture with 0.5 ug of the above cDNAtemplate. A convenient reaction mixture is 1.5-5 mM MgCl₂, 0.01% (w/v)gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primerand 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturationat 94 degree C. for 1 min; annealing at 55 degree C. for 1 min;elongation at 72 degree C. for 1 min) are performed with a Perkin-ElmerCetus automated thermal cycler. The amplified product is analyzed byagarose gel electrophoresis and the DNA band with expected molecularweight is excised and purified. The PCR product is verified to be theselected sequence by subcloning and sequencing the DNA product.

[0353] Several methods are available for the identification of the 5′ or3′ non-coding portions of the D-SLAM gene which may not be present inthe deposited clone. These methods include but are not limited to,filter probing, clone enrichment using specific probes, and protocolssimilar or identical to 5′ and 3′ “RACE” protocols which are well knownin the art. For instance, a method similar to 5′ RACE is available forgenerating the missing 5′ end of a desired full-length transcript.(Fromont-Racine et al., Nucleic Acids Res. 21(7):1683-1684 (1993).)

[0354] Briefly, a specific RNA oligonucleotide is ligated to the 5′ endsof a population of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of theD-SLAM gene of interest is used to PCR amplify the 5′ portion of theD-SLAM full-length gene. This amplified product may then be sequencedand used to generate the full length gene.

[0355] This above method starts with total RNA isolated from the desiredsource, although poly-A+ RNA can be used. The RNA preparation can thenbe treated with phosphatase if necessary to eliminate 5′ phosphategroups on degraded or damaged RNA which may interfere with the later RNAligase step. The phosphatase should then be inactivated and the RNAtreated with tobacco acid pyrophosphatase in order to remove the capstructure present at the 5′ ends of messenger RNAs. This reaction leavesa 5′ phosphate group at the 5′ end of the cap cleaved RNA which can thenbe ligated to an RNA oligonucleotide using T4 RNA ligase.

[0356] This modified RNA preparation is used as a template for firststrand cDNA synthesis using a gene specific oligonucleotide. The firststrand synthesis reaction is used as a template for PCR amplification ofthe desired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the D-SLAM gene.

Example 2 Isolation of D-SLAM Genomic Clones

[0357] A human genomic P1 library (Genomic Systems, Inc.) is screened byPCR using primers selected for the cDNA sequence corresponding to SEQ IDNO:1., according to the method described in Example 1. (See also,Sambrook.)

Example 3 Tissue Distribution of D-SLAM Polypeptides

[0358] Tissue distribution of mRNA expression of D-SLAM is determinedusing protocols for Northern blot analysis, described by, among others,Sambrook et al. For example, a D-SLAM probe produced by the methoddescribed in Example 1 is labeled with P³² using the rediprime™ DNAlabeling system (Amersham Life Science), according to manufacturer'sinstructions. After labeling, the probe is purified using CHROMASPIN-100™ column (Clontech Laboratories, Inc.), according tomanufacturer's protocol number PT1200-1. The purified labeled probe isthen used to examine various human tissues for mRNA expression.

[0359] Multiple Tissue Northern (MTN) blots containing various humantissues (H) or human immune system tissues (IM) (Clontech) are examinedwith the labeled probe using ExpressHyb™ hybridization solution(Clontech) according to manufacturer's protocol number PT1190-1.Following hybridization and washing, the blots are mounted and exposedto film at −70 degree C. overnight, and the films developed according tostandard procedures.

Example 4 Chromosomal Mapping of D-SLAM

[0360] An oligonucleotide primer set is designed according to thesequence at the 5′ end of SEQ ID NO:1. This primer preferably spansabout 100 nucleotides. This primer set is then used in a polymerasechain reaction under the following set of conditions: 30 seconds, 95degree C.; 1 minute, 56 degree C.; 1 minute, 70 degree C. This cycle isrepeated 32 times followed by one 5 minute cycle at 70 degree C. Human,mouse, and hamster DNA is used as template in addition to a somatic cellhybrid panel containing individual chromosomes or chromosome fragments(Bios, Inc). The reactions is analyzed on either 8% polyacrylamide gelsor 3.5% agarose gels. Chromosome mapping is determined by the presenceof an approximately 100 bp PCR fragment in the particular somatic cellhybrid.

Example 5 Bacterial Expression of D-SLAM

[0361] D-SLAM polynucleotide encoding a D-SLAM polypeptide invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined in Example 1, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites, such as BamHI and XbaI, at the 5′end of the primers in order to clone the amplified product into theexpression vector. For example, BamHI and XbaI correspond to therestriction enzyme sites on the bacterial expression vector pQE-9.(Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter/operator (P/O), a ribosome bindingsite (RBS), a 6-histidine tag (6-His), and restriction enzyme cloningsites.

[0362] The pQE-9 vector is digested with BamHI and XbaI and theamplified fragment is ligated into the pQE-9 vector maintaining thereading frame initiated at the bacterial RBS. The ligation mixture isthen used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) whichcontains multiple copies of the plasmid pREP4, which expresses the lacIrepressor and also confers kanamycin resistance (Kan^(r)). Transformantsare identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

[0363] Alternatively, a construct containing DNA encoding amino acidQ24-D233 of SEQ ID NO:2 can be inserted into pQE70. This constructplaces a HIS tag (6 histidines) at the C-terminus of the predictedextracellular domain of D-SLAM. Primers that can be used include a 5′primer containing a Sph restriction site, shown in bold:

[0364] GCAGCAGCATGCAAGTGCTGAGCAAAGTCGGGGGCTCGGTGCTG (SEQ ID NO:14) and a3′ primer, containing a BglII restriction site, shown in bold:

[0365] GCAGCAAGATCTATCTTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO:15).

[0366] This construct uses an ATG as a start codon contained within theSphI site, then reading into Q24 of SEQ ID NO:2, and continues untilD233 of SEQ ID NO:2. The amino acid sequence encoded by this constructis follows: (SEQ ID NO: 16)MQVLSKVGGSVLLVAARPPGFQVREAIWRSLWPSEELLATFFRGSLETLYHSRFLGRAQLHSNLSLELGPLESGDSGNFSVLMVDTRGQPWTQTLQLKVYDAVPRPVVQVFIAVERDAQPSKTCQVFLSCWAPNISEITYSWRRETTMDFGMEPHSLFTDGQVLSISLGPGDRDVAYSCIVSNPVSWDLATVTPWDSCHHEAAPGKASYKDQVLSKVGGSVLLVAARPPGFQVREAIWRSLWPSEELLATFFRGSLETLYHSRFLGRAQLHSNLSLELGPLESGDSGNFSVLMVDTRGQPWTQTLQLKVYDAVPRPVVQVFIAVERDAQPSKTCQVFLSCWAPNISEITYSWRRETTMDFGMEPHSLFTDGQVLSISLGPGDRDVAYSCIVSNPVSWDLATVTPWDSCHHEAAPGKASYKDHHHHHH.

[0367] Alternatively, a His tag can be placed on the N-terminus of thepredicted mature form containing only the extracellular domain of D-SLAM(e.g., corresponding to A23-D233 of SEQ ID NO:2). In this example, the5′ primer, containing a BamHI restriction site, indicated in bold, canbe used: (SEQ ID NO: 17) GCAGCAGGATCCGCCCAAGTGCTGAGCAAAGTCGGGGGCTCGGTGand a 3′ primer can be used: (SEQ ID NO: 18)GCAGCAAAGCTTTTAATCTTTGTAGGAGGCCTTCCCTGGTGCTGCCTC.

[0368] These primer can be used to amplify DNA encoding A23-D233, andthen the generated product can be inserted into pQE9. This constructputs a His tag on the N-terminus of the predicted mature extracellulardomain of D-SLAM. The His tag will be followed by the Gly-Ser of theBamHI site, and this will then be followed by A23 of SEQ ID NO:2. Thisconstruct will continue through D233 of SEQ ID NO:2, and will befollowed by a TAA stop codon. The amino acid sequence encoded by thisconstruct is as follows: (SEQ ID NO: 19)MRGSHHHHHHGSAQVLSKVGGSVLLVAARPPGFQVREAIWRSLWPSEELLATFFRGSLETLYHSRFLGRAQLHSNLSLELGPLESGDSGNFSVLMVDTRGQPWTQTLQLKVYDAVPRPVVQVFIAVERDAQPSKTCQVFLSCWAPNISEITYSWRRETTMDFGMEPHSLFTDGQVLSISLGPGDRDVAYSCIVSNPVSWDLATVTPWDSCHHEAAPGKASYKD.

[0369] Additionally, a mature form containing only the extracellulardomain of D-SLAM (amino acids A23 to D233 of SEQ ID NO:2) can also beinserted into an E. coli expression vector, such as pHE4 (see below). Inthis example, the 5′ primer, containing a Nde restriction site,indicated in bold, can be used:

[0370] GCAGCACATATGGCCCAAGTGCTGAGCAAAGTCG (SEQ ID NO:20) and a 3′ primercontaining an Asp718 restriction site, shown in bold, can be used:

[0371] GCAGCAGGTACCTTACTAATCTTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO:21).

[0372] Clones containing the desired constructs are grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells are grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(Isopropyl-B-D-thiogalacto pyranoside) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression.

[0373] Cells are grown for an extra 3 to 4 hours. Cells are thenharvested by centrifugation (20 mins at 6000×g). The cell pellet issolubilized in the chaotropic agent 6 Molar Guanidine HCl by stirringfor 3-4 hours at 4 degree C. The cell debris is removed bycentrifugation, and the supernatant containing the polypeptide is loadedonto a nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6 × His tag bindto the Ni-NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

[0374] Briefly, the supernatant is loaded onto the column in 6 Mguanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 Mguanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.

[0375] The purified D-SLAM protein is then renatured by dialyzing itagainst phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 bufferplus 200 mM NaCl. Alternatively, the D-SLAM protein can be successfullyrefolded while immobilized on the Ni-NTA column. The recommendedconditions are as follows: renature using a linear 6M-1M urea gradientin 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing proteaseinhibitors. The renaturation should be performed over a period of 1.5hours or more. After renaturation the proteins are eluted by theaddition of 250 mM immidazole. Immidazole is removed by a finaldialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200mM NaCl. The purified D-SLAM protein is stored at 4 degree C. or frozenat −80 degree C.

[0376] In addition to the above expression vector, the present inventionfurther includes an expression vector comprising phage operator andpromoter elements operatively linked to a D-SLAM polynucleotide, calledpHE4a. (ATCC Accession Number 209645, deposited Feb. 25, 1998.) Thisvector contains: 1) a neomycinphosphotransferase gene as a selectionmarker, 2) an E. coli origin of replication, 3) a T5 phage promotersequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence,and 6) the lactose operon repressor gene (lacIq). The origin ofreplication (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). Thepromoter sequence and operator sequences are made synthetically.

[0377] DNA can be inserted into the pHEa by restricting the vector withNdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted product ona gel, and isolating the larger fragment (the stuffer fragment should beabout 310 base pairs). The DNA insert is generated according to the PCRprotocol described in Example 1, using PCR primers having restrictionsites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer).The PCR insert is gel purified and restricted with compatible enzymes.The insert and vector are ligated according to standard protocols.

[0378] The engineered vector could easily be substituted in the aboveprotocol to express protein in a bacterial system.

Example 6 Purification of D-SLAM Polypeptide from an Inclusion Body

[0379] The following alternative method can be used to purify D-SLAMpolypeptide expressed in E coli when it is present in the form ofinclusion bodies. Unless otherwise specified, all of the following stepsare conducted at 4-10 degree C.

[0380] Upon completion of the production phase of the E. colifermentation, the cell culture is cooled to 4-10 degree C. and the cellsharvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech).On the basis of the expected yield of protein per unit weight of cellpaste and the amount of purified protein required, an appropriate amountof cell paste, by weight, is suspended in a buffer solution containing100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to ahomogeneous suspension using a high shear mixer.

[0381] The cells are then lysed by passing the solution through amicrofluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

[0382] The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4 degree C. overnight to allowfurther GuHCl extraction.

[0383] Following high speed centrifugation (30,000×g) to removeinsoluble particles, the GuHCl solubilized protein is refolded byquickly mixing the GuHCl extract with 20 volumes of buffer containing 50mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. Therefolded diluted protein solution is kept at 4 degree C. without mixingfor 12 hours prior to further purification steps.

[0384] To clarify the refolded polypeptide solution, a previouslyprepared tangential filtration unit equipped with 0.16 um membranefilter with appro priate surface area (e.g., Filtron), equilibrated with40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loadedonto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems).The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in astepwise manner. The absorbance at 280 nm of the effluent iscontinuously monitored. Fractions are collected and further analyzed bySDS-PAGE.

[0385] Fractions containing the D-SLAM polypeptide are then pooled andmixed with 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perseptive Biosystems) and weak anion (Poros CM-20, PerseptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A₂₈₀ monitoring of the effluent. Fractionscontaining the polypeptide (determined, for instance, by 16% SD S-PAGE)are then pooled.

[0386] The resultant D-SLAM polypeptide should exhibit greater than 95%purity after the above refolding and purification steps. No majorcontaminant bands should be observed from Commassie blue stained 16%SDS-PAGE gel when 5 ug of purified protein is loaded. The purifiedD-SLAM protein can also be tested for endotoxin/LPS contamination, andtypically the LPS content is less than 0.1 ng/ml according to LALassays.

Example 7 Cloning and Expression of D-SLAM in a Baculovirus ExpressionSystem

[0387] In this example, the plasmid shuttle vector pA2 is used to insertD-SLAM polynucleotide into a baculovirus to express D-SLAM. Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed byconvenient restriction sites such as BamHI, Xba I and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate a viable virus thatexpress the cloned D-SLAM polynucleotide.

[0388] Many other baculovirus vectors can be used in place of the vectorabove, such as pAc373, pVL941, and pAcIM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39 (1989).

[0389] Specifically, the D-SLAM cDNA sequence contained in the depositedclone, including the AUG initiation codon and any naturally associatedleader sequence, is amplified using the PCR protocol described inExample 1. If the naturally occurring signal sequence is used to producethe secreted protein, the pA2 vector does not need a second signalpeptide. Alternatively, the vector can be modified (pA2 GP) to include abaculovirus leader sequence, using the standard methods described inSummers et al., “A Manual of Methods for Baculovirus Vectors and InsectCell Culture Procedures,” Texas Agricultural Experimental StationBulletin No. 1555 (1987).

[0390] Fragments of D-SLAM can be expressed from the baculovirus system.For example, the predicted extracellular domain (M1-K232 of SEQ ID NO:2)can be inserted into pA2 using the primers described throughout theExample section.

[0391] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

[0392] The plasmid is digested with the corresponding restrictionenzymes and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.).

[0393] The fragment and the dephosphorylated plasmid are ligatedtogether with T4 DNA ligase. E. coli HB101 or other suitable E. colihosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.)cells are transformed with the ligation mixture and spread on cultureplates. Bacteria containing the plasmid are identified by digesting DNAfrom individual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

[0394] Five ug of a plasmid containing the polynucleotide isco-transfected with 1.0 ug of a commercially available linearizedbaculovirus DNA (“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego,Calif.), using the lipofection method described by Felgner et al., Proc.Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of BaculoGold™ virusDNA and 5 ug of the plasmid are mixed in a sterile well of a microtiterplate containing 50 ul of serum-free Grace's medium (Life TechnologiesInc., Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ulGrace's medium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mM tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27 degrees C. The transfection solution is then removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. Cultivation is then continued at 27 degrees C. forfour days.

[0395] After four days the supernatant is collected and a plaque assayis performed, as described by Summers and Smith, supra. An agarose gelwith “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10.) After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 ul of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mM dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4 degree C.

[0396] To verify the expression of the polypeptide, Sf9 cells are grownin Grace's medium supplemented with 10% heat-inactivated FBS. The cellsare infected with the recombinant baculovirus containing thepolynucleotide at a multiplicity of infection (“MOI”) of about 2. Ifradiolabeled proteins are desired, 6 hours later the medium is removedand is replaced with SF900 II medium minus methionine and cysteine(available from Life Technologies Inc., Rockville, Md.). After 42 hours,5 uCi of ³⁵S-methionine and 5 uCi ³⁵S-cysteine (available from Amersham)are added. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

[0397] Microsequencing of the amino acid sequence of the amino terminusof purified protein may be used lo determine the amino terminal sequenceof the produced D-SLAM protein.

Example 8 Expression of D-SLAM in Mammalian Cells

[0398] D-SLAM polypeptide can be expressed in a mammalian cell. Atypical mammalian expression vector contains a promoter element, whichmediates the initiation of transcription of mRNA, a protein codingsequence, and signals required for the termination of transcription andpolyadenylation of the transcript. Additional elements includeenhancers, Kozak sequences and intervening sequences flanked by donorand acceptor sites for RNA splicing. Highly efficient transcription isachieved with the early and late promoters from SV40, the long terminalrepeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the earlypromoter of the cytomegalovirus (CMV). However, cellular elements canalso be used (e.g., the human actin promoter).

[0399] Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2DHFR (ATCC37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0.Mammalian host cells that could be used include, human Hela, 293, H9 andJurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quailQC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

[0400] Alternatively, D-SLAM polypeptide can be expressed in stable celllines containing the D-SLAM polynucleotide integrated into a chromosome.The co-transfection with a selectable marker such as DHFR, gpt,neomycin, hygromycin allows the identification and isolation of thetransfected cells.

[0401] The transfected D-SLAM gene can also be amplified to expresslarge amounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful in developing cell lines that carry several hundred oreven several thousand copies of the gene of interest. (See, e.g., Alt,F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. andMa, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. andSydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selectionmarker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992).Using these markers, the mammalian cells are grown in selective mediumand the cells with the highest resistance are selected. These cell linescontain the amplified gene(s) integrated into a chromosome. Chinesehamster ovary (CHO) and NSO cells are often used for the production ofproteins.

[0402] Derivatives of the plasmid pSV2-DHFR (ATCC Accession No. 37146),the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCCAccession No.209647) contain the strong promoter (LTR) of the RousSarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447(March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell41:521-530 (1985).) Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofD-SLAM. The vectors also contain the 3′ intron, the polyadenylation andtermination signal of the rat preproinsulin gene, and the mouse DHFRgene under control of the SV40 early promoter.

[0403] Specifically, the plasmid pC6 or pC4 is digested appropriaterestriction enzymes and then dephosphorylated using calf intestinalphosphates by procedures known in the art. The vector is then isolatedfrom a 1% agarose gel.

[0404] D-SLAM polynucleotide is amplified according to the protocoloutlined in Example 1. If a naturally occurring signal sequence is usedto produce a secreted protein, the vector does not need a second signalpeptide. Alternatively, if a naturally occurring signal sequence is notused, the vector can be modified to include a heterologous signalsequence in an effort to secrete the protein from the cell. (See, e.g.,WO 96/34891.)

[0405] Specifically, the full length D-SLAM protein can be expressedfrom a mammalian vector, such as pC4, using the following primers: The5′ primer, containing a BamHI in bold, is as follows:

[0406] GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ IDNO:22) while the 3′ primer contains a Xba site shown in bold:

[0407] GCAGCATCTAGATTATGGCAGATCCTGCACAAGGGGGTTCTCTGTC (SEQ ID NO: 23).

[0408] This construct should produce a transmembrane protein that willbe expressed on the external cell surface.

[0409] Alternatively, a construct containing only the soluble portion ofD-SLAM can be made by inserting the predicted extracellular domain ofD-SLAM in pC4. For example, DNA encoding M1-K232 of SEQ ID NO:2 can bein inserted into pC4, using a 5′ primer, containing a BamHI restrictionsite shown in bold:

[0410] GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ IDNO:22) and a 3′ primer, containing a Xba restriction site shown in bold:

[0411] GCAGCATCTAGATTATTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO:24).

[0412] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel. The amplified fragmentis then digested with the same restriction enzyme and purified on a 1%agarose gel. The isolated fragment and the dephosphorylated vector arethen ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells arethen transformed and bacteria are identified that contain the fragmentinserted into plasmid pC6 or pC4 using, for instance, restriction enzymeanalysis.

[0413] Chinese hamster ovary cells lacking an active DHFR gene is usedfor transfection. Five μg of the expression plasmid pC6 or pC4 iscotransfected with 0.5 ug of the plasmid pSVneo using lipofectin(Felgner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the neo gene from Tn5 encoding an enzyme that confersresistance to a group of antibiotics including G418. The cells areseeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,the cells are trypsinized and seeded in hybridoma cloning plates(Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50ng/ml of metothrexate plus 1 mg/ml G418. After about 10-14 days singleclones are trypsinized and then seeded in 6-well petri dishes or 10 mlflasks using different concentrations of methotrexate (50 nM, 100 nM,200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations ofmethotrexate are then transferred to new 6-well plates containing evenhigher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM).The same procedure is repeated until clones are obtained which grow at aconcentration of 100-200 uM. Expression of D-SLAM is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 9 Construction of N-Terminal and/or C-Terminal Deletion Mutants

[0414] The following general approach may be used to clone a N-terminalor C-terminal deletion D-SLAM deletion mutant. Generally, twooligonucleotide primers of about 15-25 nucleotides are derived from thedesired 5′ and 3′ positions of a polynucleotide of SEQ ID NO:1. The 5′and 3′ positions of the primers are determined based on the desiredD-SLAM polynucleotide fragment. An initiation and stop codon are addedto the 5′ and 3′ primers respectively, if necessary, to express theD-SLAM polypeptide fragment encoded by the polynucleotide fragment.Preferred D-SLAM polynucleotide fragments are those encoding theN-terminal and C-terminal deletion mutants disclosed above in the“Polynucleotide and Polypeptide Fragments” section of the Specification.

[0415] Additional nucleotides containing restriction sites to facilitatecloning of the D-SLAM polynucleotide fragment in a desired vector mayalso be added to the 5′ and 3′ primer sequences. The D-SLAMpolynucleotide fragment is amplified from genomic DNA or from thedeposited cDNA clone using the appropriate PCR oligonucleotide primersand conditions discussed herein or known in the art. The D-SLAMpolypeptide fragments encoded by the D-SLAM polynucleotide fragments ofthe present invention may be expressed and purified in the same generalmanner as the full length polypeptides, although routine modificationsmay be necessary due to the differences in chemical and physicalproperties between a particular fragment and full length polypeptide.

[0416] As a means of exemplifying but not limiting the presentinvention, the polynucleotide encoding the D-SLAM polypeptide fragmentLeu-35 to Thr-276 is amplified and cloned as follows: A 5′ primer isgenerated comprising a restriction enzyme site followed by an initiationcodon in frame with the polynucleotide sequence encoding the N-terminalportion of the polypeptide fragment beginning with Leu-35. Acomplementary 3′ primer is generated comprising a restriction enzymesite followed by a stop codon in frame with the polynucleotide sequenceencoding C-terninal portion of the D-SLAM polypeptide fragment endingwith Thr-276.

[0417] The amplified polynucleotide fragment and the expression vectorare digested with restriction enzymes which recognize the sites in theprimers. The digested polynucleotides are then ligated together. TheD-SLAM polynucleotide fragment is inserted into the restrictedexpression vector, preferably in a manner which places the D-SLAMpolypeptide fragment coding region downstream from the promoter. Theligation mixture is transformed into competent E. coli cells usingstandard procedures and as described in the Examples herein. Plasmid DNAis isolated from resistant colonies and the identity of the cloned DNAconfirmed by restriction analysis, PCR and DNA sequencing.

Example 10 Protein Fusions of D-SLAM

[0418] D-SLAM polypeptides are preferably fused to other proteins. Thesefusion proteins can be used for a variety of applications. For example,fusion of D-SLAM polypeptides to His-tag, HA-tag, protein A, IgGdomains, and maltose binding protein facilitates purification. (SeeExample 5; see also EP A 394,827; Traunecker, et al., Nature 331:84-86(1988).) Similarly, fusion to IgG-1, IgG-3, and albumin increases thehalflife time in vivo. Nuclear localization signals fused to D-SLAMpolypeptides can target the protein to a specific subcellularlocalization, while covalent heterodimer or homodimers can increase ordecrease the activity of a fusion protein. Fusion proteins can alsocreate chimeric molecules having more than one function. Finally, fusionproteins can increase solubility and/or stability of the fused proteincompared to the non-fused protein. All of the types of fusion proteinsdescribed above can be made by modifying the following protocol, whichoutlines the fusion of a polypeptide to an IgG molecule, or the protocoldescribed in Example 5.

[0419] Briefly, the human Fc portion of the IgG molecule can be PCRamplified, using primers that span the 5′ and 3′ ends of the sequencedescribed below. These primers also should have convenient restrictionenzyme sites that will facilitate cloning into an expression vector,preferably a mammalian expression vector.

[0420] For example, if pC4 (Accession No. 209646) is used, the human Fcportion can be ligated into the BamHI cloning site. Note that the 3′BamHI site should be destroyed. Next, the vector containing the human Fcportion is re-restricted with BamHI, linearizing the vector, and D-SLAMpolynucleotide, isolated by the PCR protocol described in Example 1, isligated into this BamHI site. Note that the polynucleotide is clonedwithout a stop codon, otherwise a fusion protein will not be produced.

[0421] Examples of primers that can be used to amplify D-SLAMpolypeptides, such as the predicted extracellular domain of D-SLAMinclude: a 5′ primer containing a BamHI restriction site shown in bold:

[0422] GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ IDNO:22) and a 3′ primer, containing a Xba restriction site shown in bold:

[0423] GCAGCATCTAGAATCTTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO:25).

[0424] Using these primers, the construct will express the predictedextracellular domain of D-SLAM fused to Fc in pC4.

[0425] If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.)

[0426] Human IgG Fc region:GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACC (SEQ ID NO:4) TGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

[0427] Alternatively, this same region can be inserted in pA2, creatinga fusion of Fc with the predicted extracellular domain of D-SLAM.Examples of primer that can be used to amplify the predictedextracellular domain of D-SLAM include: a 5′ primer containing a BamHIrestriction site shown in bold:

[0428] GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ IDNO:22) and a 3′ primer, containing a Xba restriction site shown in bold:

[0429] GCAGCATCTAGAATCTTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO:25).

[0430] This construct will express the predicted extracellular domain ofD-SLAM fused to Fc in pA2.

[0431] These two above constructs were expressed in their appropriatehost cells (e.g., pC4 in a mammalian system, while pA2 in a baculovirussystem), both systems produced a truncated protein, lacking thepredicted signal sequence and beginning with A23 of SEQ ID NO:2. Thus,as was predicted, both baculovirus and mammalian systems process D-SLAMto amino acid A23 of SEQ ID NO:2. However, due to differences inglycosylation, the baculovirus system produces a protein, under reducingconditions, migrating at around 60 kD, while the mammalian systemgenerates a protein migrating at around 55 kD.

[0432] Additionally, fusion proteins of D-SLAM polypeptides, includingthe predicted extracellular domain or the mature form of D-SLAM, can befused to FLAG for mammalian cell expression. For example, theextracellular domain can be amplifed using the following primers: a 5′primer containing a BamHI restriction site shown in bold:

[0433] GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ IDNO:22) and a 3′ primer, containing a Xba restriction site shown in bold:

[0434] GCAGCATCTAGATTACTTGTCATCGTCGTCCTTGTAGTCATCTTTGTAGGAGGCCTTCCCTGGTGCTG (SEQ ID NO:26).

Example 11 Production of an Antibody

[0435] a) Hybridoma Technology

[0436] The antibodies of the present invention can be prepared by avariety of methods. (See, Current Protocols, Chapter 2.) As one exampleof such methods, cells expressing D-SLAM is administered to an animal toinduce the production of sera containing polyclonal antibodies. In apreferred method, a preparation of D-SLAM protein is prepared andpurified to render it substantially free of natural contaminants. Such apreparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

[0437] In the most preferred method, the antibodies of the presentinvention are monoclonal antibodies (or protein binding fragmentsthereof). Such monoclonal antibodies can be prepared using hybridomatechnology. (Köhler et al., Nature 256:495 (1975); Köhler et al., Eur.J. Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas,Elsevier, N.Y., pp. 563-681 (1981).) In general, such procedures involveimmunizing an animal (preferably a mouse) with D-SLAM polypeptide or,more preferably, with a secreted D-SLAM polypeptide-expressing cell.Such cells may be cultured in any suitable tissue culture medium;however, it is preferable to culture cells in Earle's modified Eagle'smedium supplemented with 10% fetal bovine serum (inactivated at about 56degree C.), and supplemented with about 10 g/l of nonessential aminoacids, about 1,000 U/ml of penicillin, and about 100 ug/ml ofstreptomycin.

[0438] The splenocytes of such mice are extracted and fused with asuitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention; however, it ispreferable to employ the parent myeloma cell line (SP20), available fromthe ATCC. After fusion, the resulting hybridoma cells are selectivelymaintained in HAT medium, and then cloned by limiting dilution asdescribed by Wands et al. (Gastroenterology 80:225-232 (1981).) Thehybridoma cells obtained through such a selection are then assayed toidentify clones which secrete antibodies capable of binding the D-SLAMpolypeptide.

[0439] Alternatively, additional antibodies capable of binding to D-SLAMpolypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodywhich binds to a second antibody. In accordance with this method,protein specific antibodies are used to immunize an animal, preferably amouse. The splenocytes of such an animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify cloneswhich produce an antibody whose ability to bind to the D-SLAMprotein-specific antibody can be blocked by D-SLAM. Such antibodiescomprise anti-idiotypic antibodies to the D-SLAM protein-specificantibody and can be used to immunize an animal to induce formation offurther D-SLAM protein-specific antibodies.

[0440] It will be appreciated that Fab and F(ab′)2 and other fragmentsof the antibodies of the present invention may be used according to themethods disclosed herein. Such fragments are typically produced byproteolytic cleavage, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)2 fragments). Alternatively,secreted D-SLAM protein-binding fragments can be produced through theapplication of recombinant DNA technology or through syntheticchemistry.

[0441] For in vivo use of antibodies in humans, it may be preferable touse “humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. (See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).)

[0442] b) Isolation of Antibody Fragments Directed Against D-SLAM from aLibrary of scFvs

[0443] Naturally occuring V-genes isolated from human PBLs areconstructed into a large library of antibody fragments which containreactivities against D-SLAM to which the donor may or may not have beenexposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein in itsentirety by reference).

[0444] Rescue of the Library

[0445] A library of scFvs is constructed from the RNA of human PBLs asdescribed in WO92/01047. To rescue phage displaying antibody fragments,approximately 10⁹ E. coli harbouring the phagemid are used to inoculate50 ml of 2×TY containing 1% glucose and 100 ug/ml of ampicillin(2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of thisculture is used to innoculate 50 ml of 2×TY-AMP-GLU, 2×10⁸ TU of deltagene 3 helper (M13 delta gene III, see WO92/01047) are added and theculture incubated at 37 degree C. for 45 minutes without shaking andthen at 37 degree C. for 45 minutes with shaking. The culture iscentrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended in 2liters of of 2×TY containing 100 ug/ml ampicillin and 50 ug/ml kanamycinand grown overnight. Phage are prepared as described in WO92/01047.

[0446] M13 delta gene III is prepared as follows: M13 delta gene IIIhelper phage does not encode gene III protein, hence the phage(mid)displaying antibody fragments have a greater avidity of binding toantigen. Infectious M13 delta gene III particles are made by growing thehelper phage in cells harbouring a pUC19 derivative supplying the wildtype gene III protein during phage morphogenesis. The culture isincubated for 1 hour at 37 degree C. without shaking and then for afurther hour at 37 degree C. with shaking. Cells are spun down(IEC-Centra 8, 4000 revs/min for 10 min), resuspended in 300 ml 2×TYbroth containing 100 ug ampicillin/ml and 25 ug kanamycin/ml(2×TY-AMP-KAN) and grown overnight, shaking at 37° C. Phage particlesare purified. and concentrated from the culture medium by twoPEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS andpassed through a 0.45 um filter (Minisart NML; Sartorius) to give afinal concentration of approximately 10¹³ transducing units/nil(ampicillin-resistant clones).

[0447] Panning of the Library

[0448] Immunotubes (Nunc) are coated overnight in PBS with 4 ml ofeither 100 ug/ml or 10 ug/ml of a polypeptide of the present invention.Tubes are blocked with 2% Marvel-PBS for 2 hours at 37 degree C. andthen washed 3 times in PBS. Approximately 10¹³ TU of phage is applied tothe tube and incubated for 30 minutes at room temperature tumbling on anover and under turntable and then left to stand for another 1.5 hours.Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS.Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15minutes on an under and over turntable after which the solution isimmediately neutralized with 0.5 ml of 1.0 M Tris-HCl, pH 7.4. Phage arethen used to infect 10 ml of mid-log E. coli TG1 by incubating elutedphage with bacteria for 30 minutes at 37 degree C. The E. coli are thenplated on TYE plates containing 1% glucose and 100 ug/ml ampicillin. Theresulting bacterial library is then rescued with delta gene 3 helperphage as described above to prepare phage for a subsequent round ofselection. This process is then repeated for a total of 4 rounds ofaffinity purification with tube-washing increased to 20 times with PBS,0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

[0449] Characterization of Binders

[0450] Eluted phage from the 3rd and 4th rounds of selection are used toinfect E. coli HB 2151 and soluble scFv is produced (Marks, et al.,1991) from single colonies for assay. ELISAs are performed withmicrotitre plates coated with either 10 pg/ml of the polypeptide of thepresent invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISAare further characterized by PCR fingerprinting (see e.g., WO92/01047)and then by sequencing.

Example 12 Production Of D-SLAM-Protein for High-Throughput ScreeningAssays

[0451] The following protocol produces a supernatant containing D-SLAMpolypeptide to be tested. This supernatant can then be used in theScreening Assays described in Examples 14-21.

[0452] First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stocksolution (1 mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516FBlowhittaker) for a working solution of 50 ug/ml. Add 200 ul of thissolution to each well (24 well plates) and incubate at RT for 20minutes. Be sure to distribute the solution over each well (note: a12-channel pipetter may be used with tips on every other channel).Aspirate off the Poly-D-Lysine solution and rinse with 1 ml PBS(Phosphate Buffered Saline). The PBS should remain in the well untiljust prior to plating the cells and plates may be poly-lysine coated inadvance for up to two weeks.

[0453] Plate 293T cells (do not carry cells past P+20) at 2¹⁰ ⁵cells/well in 0.5 ml DMEM (Dulbecco's Modified Eagle Medium) (with 4.5G/L glucose and L-glutamine (12-604F Biowhittaker))/10% heat inactivatedFBS (14-503F Biowhittaker)/1× Penstrep (17-602E Biowhittaker). Let thecells grow overnight.

[0454] The next day, mix together in a sterile solution basin: 300 ulLipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter,aliquot approximately 2 ug of an expression vector containing apolynucleotide insert, produced by the methods described in Examples8-10, into an appropriately labeled 96-well round bottom plate. With amulti-channel pipetter, add 50 ul of the Lipofectamine/Optimem I mixtureto each well. Pipette up and down gently to mix. Incubate at RT 15-45minutes. After about 20 minutes, use a multi-channel pipetter to add 150ul Optimem I to each well. As a control, one plate of vector DNA lackingan insert should be transfected with each set of transfections.

[0455] Preferably, the transfection should be performed by tag-teamingthe following tasks. By tag-teaming, hands on time is cut in half, andthe cells do not spend too much time on PBS. First, person A aspiratesoff the media from four 24-well plates of cells, and then person Brinses each well with 0.5-1 ml PBS. Person A then aspirates off PBSrinse, and person B, using a 12-channel pipetter with tips on everyother channel, adds the 200 ul of DNA/Lipofectamine/Optimem I complex tothe odd wells first, then to the even wells, to each row on the 24-wellplates. Incubate at 37 degree C. for 6 hours.

[0456] While cells are incubating, prepare appropriate media, either 1%BSA in DMEM with 1× penstrep, or HGS CHO-5 media (116.6 mg/L of CaCl2(anhyd); 0.00130 mg/L CuSO₄—5H₂O; 0.050 mg/L of Fe(NO₃)₃—9H₂O; 0.417mg/L of FeSO₄—7H₂O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl₂; 48.84 mg/Lof MgSO₄; 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO₃; 62.50 mg/L ofNaH₂PO₄—H₂O; 71.02 mg/L of Na₂HPO4; 0.4320 mg/L of ZnSO₄—7H₂O; 0.002mg/L of Arachidonic Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L ofDL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L ofLinolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid;0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L ofPluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551mg/L of D-Glucose; 130.85 mg/ml of L-Alanine; 147.50 mg/ml ofL-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H₂O; 6.65 mg/ml of L-AsparticAcid; 29.56 mg/ml of L-Cystine-2HCL—H₂O; 31.29 mg/ml of L-Cystine-2HCL;7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/mlof Glycine; 52.48 mg/ml of L-Histidine-HCL-H₂O; 106.97 mg/ml ofL-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL;32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0 mg/mlof L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine;19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H₂O; and99.65 mg/ml of L-Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-CaPantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid;15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L ofPyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin;3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; 0.680 mg/L ofVitamin B₁₂; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L ofSodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 uM of Ethanolamine;0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrincomplexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrincomplexed with Oleic Acid; 10 mg/L of Methyl-B-Cyclodextrin complexedwith Retinal Acetate. Adjust osmolarity to 327 mOsm) with 2 mM glutamineand 1× penstrep. (BSA (81-068-3 Bayer) 100 gm dissolved in 1L DMEM for a10% BSA stock solution). Filter the media and collect 50 ul forendotoxin assay in 15 ml polystyrene conical.

[0457] The transfection reaction is terminated, preferably bytag-teaming, at the end of the incubation period. Person A aspirates offthe transfection media, while person B adds 1.5 ml appropriate media toeach well. Incubate at 37 degree C. for 45 or 72 hours depending on themedia used: 1% BSA for 45 hours or CHO-5 for 72 hours.

[0458] On day four, using a 300 ul multichannel pipetter, aliquot 600 ulin one 1 ml deep well plate and the remaining supernatant into a 2 mldeep well. The supernatants from each well can then be used in theassays described in Examples 14-21.

[0459] It is specifically understood that when activity is obtained inany of the assays described below using a supernatant, the activityoriginates from either the D-SLAM polypeptide directly (e.g., as asecreted protein) or by D-SLAM inducing expression of other proteins,which are then secreted into the supernatant. Thus, the inventionfurther provides a method of identifying the protein in the supernatantcharacterized by an activity in a particular assay.

Example 13 Construction of GAS Reporter Construct

[0460] One signal transduction pathway involved in the differentiationand proliferation of cells is called the Jaks-STATs pathway. Activatedproteins in the Jaks-STATs pathway bind to gamma activation site “GAS”elements or interferon-sensitive responsive element (“ISRE”), located inthe promoter of many genes. The binding of a protein to these elementsalter the expression of the associated gene.

[0461] GAS and ISRE elements are recognized by a class of transcriptionfactors called Signal Transducers and Activators of Transcription, or“STATs.” There are six members of the STATs family. Stat1 and Stat3 arepresent in many cell types, as is Stat2 (as response to IFN-alpha iswidespread). Stat4 is more restricted and is not in many cell typesthough it has been found in T helper class I, cells after treatment withIL-12. Stat5 was originally called mammary growth factor, but has beenfound at higher concentrations in other cells including myeloid cells.It can be activated in tissue culture cells by many cytokines.

[0462] The STATs are activated to translocate from the cytoplasm to thenucleus upon tyrosine phosphorylation by a set of kinases known as theJanus Kinase (“Jaks”) family. Jaks represent a distinct family ofsoluble tyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. Thesekinases display significant sequence similarity and are generallycatalytically inactive in resting cells.

[0463] The Jaks are activated by a wide range of receptors summarized inthe Table below. (Adapted from review by Schidler and Darnell, Ann. Rev.Biochem. 64:621-51 (1995).) A cytokine receptor family, capable ofactivating Jaks, is divided into two groups: (a) Class 1 includesreceptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15,Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b)Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share aconserved cysteine motif (a set of four conserved cysteines and onetryptophan) and a WSXWS motif (a membrane proxial region encodingTrp-Ser-Xxx-Trp-Ser (SEQ ID NO:5)).

[0464] Thus, on binding of a ligand to a receptor, Jaks are activated,which in turn activate STATs, which then translocate and bind to GASelements. This entire process is encompassed in the Jaks-STATs signaltransduction pathway.

[0465] Therefore, activation of the Jaks-STATs pathway, reflected by thebinding of the GAS or the ISRE element, can be used to indicate proteinsinvolved in the proliferation and differentiation of cells. For example,growth factors and cytokines are known to activate the Jaks-STATspathway. (See Table below.) Thus, by using GAS elements linked toreporter molecules, activators of the Jaks-STATs pathway can beidentified. JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS (elements) or ISREIFN family IFN-a/B + + − − 1, 2, 3 ISRE IFN-g + + − 1 GAS (IRF1 > Lys6 >IFP) I1-10 + ? ? − 1, 3 gp130 family IL-6 (Pleiotrohic) + + + ? 1, 3 GAS(IRF1 > Lys6 > IFP) I1-11 (Pleiotrohic) ? + ? ? 1, 3 OnM (Pleiotrohic)? + + ? 1, 3 LIF (Pleiotrohic) ? + + ? 1, 3 CNTF (Pleiotrohic) −/+ + + ?1, 3 G-CSF (Pleiotrohic) ? + ? ? 1, 3 IL-12 (Pleiotrohic) + − + + 1, 3g-C family IL-2 (lymphocytes) − + − + 1, 3, 5 GAS IL-4 (lymph/myeloid)− + − + 6 GAS (IRF1 = IFP >> Ly6)(IgH) IL-7 (lymphocytes) − + − + 5 GASIL-9 (lymphocytes) − + − + 5 GAS IL-13 (lymphocyte) − + ? ? 6 GAS IL-15? + ? + 5 GAS gp140 family IL-3 (myeloid) − − + − 5 GAS (IRF1 > IFP >>Ly6) IL-5 (myeloid) − − + − 5 GAS GM-CSF (myeloid) − − + − 5 GAS Growthhormone family GH ? − + − 5 PRL ? +/− + − 1, 3, 5 EPO ? − + − 5 GAS (B −CAS > IRF1 = IFP >> Ly6) Receptor Tyrosine Kinases EGF ? + + − 1, 3 GAS(IRF1) PDGF ? + + − 1, 3 CSF-1 ? + + − 1, 3 GAS (not IRF1)

[0466] To construct a synthetic GAS containing promoter element, whichis used in the Biological Assays described in Examples 14-15, a PCRbased strategy is employed to generate a GAS-SV40 promoter sequence. The5′ primer contains four tandem copies of the GAS binding site found inthe IRF1 promoter and previously demonstrated to bind STATs uponinduction with a range of cytokines (Rothman et al., Immunity 1:457-468(1994).), although other GAS or ISRE elements can be used instead. The5′ primer also contains 18 bp of sequence complementary to the SV40early promoter sequence and is flanked with an XhoI site. The sequenceof the 5′ primer is: (SEQ ID NO: 6)5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3′.

[0467] The downstream primer is complementary to the SV40 promoter andis flanked with a Hind III site: 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′(SEQID NO:7).

[0468] PCR amplification is performed using the SV40 promoter templatepresent in the B-gal:promoter plasmid obtained from Clontech. Theresulting PCR fragment is digested with XhoI/Hind III and subcloned intoBLSK2−. (Stratagene.) Sequencing with forward and reverse primersconfirms that the insert contains the following sequence: (SEQ ID NO: 8)5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3′.

[0469] With this GAS promoter element linked to the SV40 promoter, aGAS:SEAP2 reporter construct is next engineered. Here, the reportermolecule is a secreted alkaline phosphatase, or “SEAP.” Clearly,however, any reporter molecule can be instead of SEAP, in this or in anyof the other Examples. Well known reporter molecules that can be usedinstead of SEAP include chloramphenicol acetyltransferase (CAT),luciferase, alkaline phosphatase, B-galactosidase, green fluorescentprotein (GFP), or any protein detectable by an antibody.

[0470] The above sequence confirmed synthetic GAS-SV40 promoter elementis subcloned into the pSEAP-Promoter vector obtained from Clontech usingHindIII and XhoI, effectively replacing the SV40 promoter with theamplified GAS:SV40 promoter element, to create the GAS-SEAP vector.However, this vector does not contain a neomycin resistance gene, andtherefore, is not preferred for mammalian expression systems.

[0471] Thus, in order to generate mammalian stable cell lines expressingthe GAS-SEAP reporter, the GAS-SEAP cassette is removed from theGAS-SEAP vector using SalI and NotI, and inserted into a backbone vectorcontaining the neomycin resistance gene, such as pGFP-1 (Clontech),using these restriction sites in the multiple cloning site, to createthe GAS-SEAP/Neo vector. Once this vector is transfected into mammaliancells, this vector can then be used as a reporter molecule for GASbinding as described in Examples 14-15.

[0472] Other constructs can be made using the above description andreplacing GAS with a different promoter sequence. For example,construction of reporter molecules containing NFK-B and EGR promotersequences are described in Examples 16 and 17. However, many otherpromoters can be substituted using the protocols described in theseExamples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can besubstituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB,I1-2/NFAT, or NF-KB/GAS). Similarly, other cell lines can be used totest reporter construct activity, such as HELA (epithelial), HUVEC(endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aortic), orCardiomyocyte.

Example 14 High-Throughput Screening Assay for T-cell Activity

[0473] The following protocol is used to assess T-cell activity ofD-SLAM by determining whether D-SLAM supernatant proliferates and/ordifferentiates T-cells. T-cell activity is assessed using theGAS/SEAP/Neo construct produced in Example 13. Thus, factors thatincrease SEAP activity indicate the ability to activate the Jaks-STATSsignal transduction pathway. The T-cell used in this assay is JurkatT-cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCCAccession No. CRL-1552) and Molt-4 cells (ATCC Accession No. CRL-1582)cells can also be used.

[0474] Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In orderto generate stable cell lines, approximately 2 million Jurkat cells aretransfected with the GAS-SEAP/neo vector using DMRIE-C (LifeTechnologies)(transfection procedure described below). The transfectedcells are seeded to a density of approximately 20,000 cells per well andtransfectants resistant to 1 mg/ml genticin selected. Resistant coloniesare expanded and then tested for their response to increasingconcentrations of interferon gamma. The dose response of a selectedclone is demonstrated.

[0475] Specifically, the following protocol will yield sufficient cellsfor 75 wells containing 200 ul of cells. Thus, it is either scaled up,or performed in multiple to generate sufficient cells for multiple 96well plates. Jurkat cells are maintained in RPMI+ 10% serum with 1%Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ug ofplasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul ofDMRIE-C and incubate at room temperature for 15-45 mins.

[0476] During the incubation period, count cell concentration, spin downthe required number of cells (10⁷ per transfection), and resuspend inOPTI-MEM to a final concentration of 10⁷ cells/ml. Then add 1 ml of1×10⁷ cells in OPTI-MEM to T25 flask and incubate at 37 degree C. for 6hrs. After the incubation, add 10 ml of RPMI+ 15% serum.

[0477] The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI+10% serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treatedwith supernatants containing D-SLAM polypeptides or D-SLAM inducedpolypeptides as produced by the protocol described in Example 12.

[0478] On the day of treatment with the supernatant, the cells should bewashed and resuspended in fresh RPMI+ 10% serum to a density of 500,000cells per ml. The exact number of cells required will depend on thenumber of supernatants being screened. For one 96 well plate,approximately 10 million cells (for 10 plates, 100 million cells) arerequired.

[0479] Transfer the cells to a triangular reservoir boat, in order todispense the cells into a 96 well dish, using a 12 channel pipette.Using a 12 channel pipette, transfer 200 ul of cells into each well(therefore adding 100,000 cells per well).

[0480] After all the plates have been seeded, 50 ul of the supernatantsare transferred directly from the 96 well plate containing thesupernatants into each well using a 12 channel pipette. In addition, adose of exogenous interferon gamma (0.1, 1.0, 10 ng) is added to wellsH9, H10, and H11 to serve as additional positive controls for the assay.

[0481] The 96 well dishes containing Jurkat cells treated withsupernatants are placed in an incubator for 48 hrs (note: this time isvariable between 48-72 hrs). 35 ul samples from each well are thentransferred to an opaque 96 well plate using a 12 channel pipette. Theopaque plates should be covered (using sellophene covers) and stored at−20 degree C. until SEAP assays are performed according to Example 18.The plates containing the remaining treated cells are placed at 4 degreeC. and serve as a source of material for repeating the assay on aspecific well if desired.

[0482] As a positive control, 100 Unit/ml interferon gamma can be usedwhich is known to activate Jurkat T cells. Over 30 fold induction istypically observed in the positive control wells.

Example 15 High-Throughput Screening Assay

[0483] Identifying Myeloid Activity

[0484] The following protocol is used to assess myeloid activity ofD-SLAM by determining whether D-SLAM proliferates and/or differentiatesmyeloid cells. Myeloid cell activity is assessed using the GAS/SEAP/Neoconstruct produced in Example 13. Thus, factors that increase SEAPactivity indicate the ability to activate the Jaks-STATS signaltransduction pathway. The mycloid cell used in this assay is U937, apre-monocyte cell line, although TF-1, HL60, or KG1 can be used.

[0485] To transiently transfect U937 cells with the GAS/SEAP/Neoconstruct produced in Example 13, a DEAE-Dextran method (Kharbanda et.al., 1994, Cell Growth & Differentiation, 5:259-265) is used. First,harvest 2×10e7 U937 cells and wash with PBS. The U937 cells are usuallygrown in RPMI 1640 medium containing 10% heat-inactivated fetal bovineserum (FBS) supplemented with 100 units/ml penicillin and 100 mg/mlstreptomycin.

[0486] Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffercontaining 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mMNaCl, 5 mM KCl, 375 uM Na₂HPO₄.7H20, 1 mM MgCl₂, and 675 uM CaCl₂.Incubate at 37 degree C. for 45 min:

[0487] Wash the cells with RPMI 1640 medium containing 10% FBS and thenresuspend in 10 ml complete medium and incubate at 37 degree C. for 36hr.

[0488] The GAS-SEAP/U937 stable cells are obtained by growing the cellsin 400 ug/ml G418. The G418-free medium is used for routine growth butevery one to two months, the cells should be re-grown in 400 ug/ml G418for couple of passages.

[0489] These cells are tested by harvesting 1×10⁸ cells (this is enoughfor ten 96-well plates assay) and wash with PBS. Suspend the cells in200 ml above described growth medium, with a final density of 5×10⁵cells/ml. Plate 200 ul cells per well in the 96-well plate (or 1×10⁵cells/well).

[0490] Add 50 ul of the supernatant prepared by the protocol describedin Example 12. Incubate at 37 degee C. for 48 to 72 hr. As a positivecontrol, 100 Unit/ml interferon gamma can be used which is known toactivate U937 cells. Over 30 fold induction is typically observed in thepositive control wells. SEAP assay the supernatant according to theprotocol described in Example 18.

Example 16 High-Throughput Screening Assay Identifying Neuronal Activity

[0491] When cells undergo differentiation and proliferation, a group ofgenes are activated through many different signal transduction pathways.One of these genes, EGR1 (early growth response gene 1), is induced invarious tissues and cell types upon activation. The promoter of EGR1 isresponsible for such induction. Using the EGR1 promoter linked toreporter molecules, activation of cells can be assessed by D-SLAM.

[0492] Particularly, the following protocol is used to assess neuronalactivity in PC12 cell lines. PC12 cells (rat phenochromocytoma cells)are known to proliferate and/or differentiate by activation with anumber of mitogens, such as TPA (tetradecanoyl phorbol acetate), NGF(nerve growth factor), and EGF (epidennal growth factor). The EGR1 geneexpression is activated during this treatment. Thus, by stablytransfecting PC12 cells with a construct containing an EGR promoterlinked to SEAP reporter, activation of PC12 cells by D-SLAM can beassessed.

[0493] The EGR/SEAP reporter construct can be assembled by the followingprotocol. The EGR-1 promoter sequence (−633 to +1) (Sakamoto K et al.,Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNAusing the following primers: (SEQ ID NO: 9)5′ GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3′ (SEQ ID NO: 10)5′ GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3′.

[0494] Using the GAS:SEAP/Neo vector produced in Example 13, EGR1amplified product can then be inserted into this vector. Linearize theGAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing theGAS/SV40 stuffer. Restrict the EGR1 amplified product with these sameenzymes. Ligate the vector and the EGR1 promoter.

[0495] To prepare 96 well-plates for cell culture, two mls of a coatingsolution (1:30 dilution of collagen type I (Upstate Biotech Inc.Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cmplate or 50 ml per well of the 96-well plate, and allowed to air dry for2 hr.

[0496] PC12 cells are routinely grown in RPMI-1640 medium (BioWhittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat. #12449-78P), 5% heat-inactivated fetal bovine serum (FBS) supplementedwith 100 units/ml penicillin and 100 ug/ml streptomycin on a precoated10 cm tissue culture dish. One to four split is done every three to fourdays. Cells are removed from the plates by scraping and resuspended withpipetting up and down for more than 15 times.

[0497] Transfect the EGR/SEAP/Neo construct into PC12 using theLipofectamine protocol described in Example 12. EGR-SEAP/PC12 stablecells are obtained by growing the cells in 300 ug/ml G418. The G418-freemedium is used for routine growth but every one to two months, the cellsshould be re-grown in 300 ug/ml G418 for couple of passages.

[0498] To assay for neuronal activity, a 10 cm plate with cells around70 to 80% confluent is screened by removing the old medium. Wash thecells once with PBS (Phosphate buffered saline). Then starve the cellsin low serum medium (RPMI-1640 containing 1% horse serum and 0.5% FBSwith antibiotics) overnight.

[0499] The next morning, remove the medium and wash the cells with PBS.Scrape off the cells from the plate, suspend the cells well in 2 ml lowserum medium. Count the cell number and add more low serum medium toreach final cell density as 5×10⁵ cells/ml.

[0500] Add 200 ul of the cell suspension to each well of 96-well plate(equivalent to 1×105 cells/well). Add 50 ul supernatant produced byExample 12, 37 degree C. for 48 to 72 hr. As a positive control, agrowth factor known to activate PC12 cells through EGR can be used, suchas 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold inductionof SEAP is typically seen in the positive control wells. SEAP assay thesupernatant according to Example 18.

Example 17 High-Throughput Screening Assay for T-cell Activity

[0501] NF-KB (Nuclear Factor KB) is a transcription factor activated bya wide variety of agents including the inflammatory cytokines IL-1 andTNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposureto LPS or thrombin, and by expression of certain viral gene products. Asa transcription factor, NF-KB regulates the expression of genes involvedin immune cell activation, control of apoptosis (NF-KB appears to shieldcells from apoptosis), B and T-cell development, anti-viral andantimicrobial responses, and multiple stress responses.

[0502] In non-stimulated conditions, NF-KB is retained in the cytoplasmwith I-KB (Inhibitor KB). However, upon stimulation, I-KB isphosphorylated and degraded, causing NF-KB to shuttle to the nucleus,thereby activating transcription of target genes. Target genes activatedby NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.

[0503] Due to its central role and ability to respond to a range ofstimuli, reporter constructs utilizing the NF-KB promoter element areused to screen the supernatants produced in Example 12. Activators orinhibitors of NF-KB would be useful in treating diseases. For example,inhibitors of NF-KB could be used to treat those diseases related to theacute or chronic activation of NF-KB, such as rheumatoid arthritis.

[0504] To construct a vector containing the NF-KB promoter element, aPCR based strategy is employed. The upstream primer contains four tandemcopies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO:11), 18 bp ofsequence complementary to the 5′ end of the SV40 early promotersequence, and is flanked with an XhoI site: (SEQ ID NO: 12)5′:GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCCTGCCATCTCAATTAG:3′.

[0505] The downstream primer is complementary to the 3′ end of the SV40promoter and is flanked with a Hind III site:5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO: 7)

[0506] PCR amplification is performed using the SV40 promoter templatepresent in the pB-gal:promoter plasmid obtained from Clontech. Theresulting PCR fragment is digested with XhoI and Hind III and subclonedinto BLSK2-. (Stratagene) Sequencing with the T7 and T3 primers confirmsthe insert contains the following sequence: (SEQ ID NO: 13)5′:CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGC AAAAAGCTT:3′.

[0507] Next, replace the SV40 minimal promoter element present in thepSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40 fragment usingXhoI and HindIII. However, this vector does not contain a neomycinresistance gene, and therefore, is not preferred for mammalianexpression systems.

[0508] In order to generate stable mammalian cell lines, theNF-KB/SV40/SEAP cassette is removed from the above NF-KB/SEAP vectorusing restriction enzymes SalI and NotI, and inserted into a vectorcontaining neomycin resistance. Particularly, the NF-KB/SV40/SEAPcassette was inserted into pGFP-1 (Clontech), replacing the GFP gene,after restricting pGFP-1 with SalI and NotI.

[0509] Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cellsare created and maintained according to the protocol described inExample 14. Similarly, the method for assaying supernatants with thesestable Jurkat T-cells is also described in Example 14. As a positivecontrol, exogenous TNF alpha (0.1, 1, 10 ng) is added to wells H9, H10,and H11, with a 5-10 fold activation typically observed.

Example 18 Assay for SEAP Activity

[0510] As a reporter molecule for the assays described in Examples14-17, SEAP activity is assayed using the Tropix Phospho-light Kit (Cat.BP-400) according to the following general procedure. The TropixPhospho-light Kit supplies the Dilution, Assay, and Reaction Buffersused below.

[0511] Prime a dispenser with the 2.5× Dilution Buffer and dispense 15ul of 2.5× dilution buffer into Optiplates containing 35 ul of asupernatant. Seal the plates with a plastic sealer and incubate at 65degree C. for 30 min. Separate the Optiplates to avoid uneven heating.

[0512] Cool the samples to room temperature for 15 minutes. Empty thedispenser and prime with the Assay Buffer. Add 50 μl Assay Buffer andincubate at room temperature 5 min. Empty the dispenser and prime withthe Reaction Buffer (see the table below). Add 50 ul Reaction Buffer andincubate at room temperature for 20 minutes. Since the intensity of thechemiluminescent signal is time dependent, and it takes about 10 minutesto read 5 plates on luminometer, one should treat 5 plates at each timeand start the second set 10 minutes later.

[0513] Read the relative light unit in the luminometer. Set H12 asblank, and print the results. An increase in chemiluminescence indicatesreporter activity. Reaction Buffer Formulation: # of plates Rxn bufferdiluent (ml) CSPD (ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 415 85 4.25 16 90 4.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 1155.75 22 120 6 23 125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28150 7.5 29 155 7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 935 185 9.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41215 10.75 42 220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12 47245 12.25 48 250 12.5 49 255 12.75 50 260 13

Example 19 High-Throughput Screening Assay Identifying Changes in SmallMolecule Concentration and Membrane Permeability

[0514] Binding of a ligand to a receptor is known to alter intracellularlevels of small molecules, such as calcium, potassium, sodium, and pH,as well as alter membrane potential. These alterations can be measuredin an assay to identify supernatants which bind to receptors of aparticular cell. Although the following protocol describes an assay forcalcium, this protocol can easily be modified to detect changes inpotassium, sodium, pH, membrane potential, or any other small moleculewhich is detectable by a fluorescent probe.

[0515] The following assay uses Fluorometric Imaging Plate Reader(“FLIPR”) to measure changes in fluorescent molecules (Molecular Probes)that bind small molecules. Clearly, any fluorescent molecule detecting asmall molecule can be used instead of the calcium fluorescent molecule,fluo-3, used here.

[0516] For adherent cells, seed the cells at 10,000-20,000 cells/well ina Co-star black 96-well plate with clear bottom. The plate is incubatedin a CO₂ incubator for 20 hours. The adherent cells are washed two timesin Biotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution)leaving 100 ul of buffer after the final wash.

[0517] A stock solution of 1 mg/ml fluo-3 is made in 10% pluronic acidDMSO. To load the cells with fluo-3, 50 ul of 12 ug/ml fluo-3 is addedto each well. The plate is incubated at 37 degree C. in a CO₂ incubatorfor 60 min. The plate is washed four times in the Biotek washer withHBSS leaving 100 ul of buffer.

[0518] For non-adherent cells, the cells are spun down from culturemedia. Cells are re-suspended to 2-5×10⁶ cells/ml with HBSS in a 50-mlconical tube. 4 ul of 1 mg/ml fluo-3 solution in 10% pluronic acid DMSOis added to each ml of cell suspension. The tube is then placed in a 37degree C. water bath for 30-60 min. The cells are washed twice withHBSS, resuspended to 1×10⁶ cells/ml, and dispensed into a microplate,100 ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plateis then washed once in Denley CellWash with 200 ul, followed by anaspiration step to 100 ul final volume.

[0519] For a non-cell based assay, each well contains a fluorescentmolecule, such as fluo-3. The supernatant is added to the well, and achange in fluorescence is detected.

[0520] To measure the fluorescence of intracellular calcium, the FLIPRis set for the following parameters: (1) System gain is 300-800 mW; (2)Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul.Increased emission at 530 nm indicates an extracellular signaling eventcaused by the a molecule, either D-SLAM or a molecule induced by D-SLAM,which has resulted in an increase in the intracellular Ca⁺⁺concentration.

Example 20 High-Throughput Screening Assay Identifying Tyrosine KinaseActivity

[0521] The Protein Tyrosine Kinases (PTK) represent a diverse group oftransmembrane and cytoplasmic kinases. Within the Receptor ProteinTyrosine Kinase RPTK) group are receptors for a range of mitogenic andmetabolic growth factors including the PDGF, FGF, EGF, NGF, HGF andInsulin receptor subfamilies. In addition there are a large family ofRPTKs for which the corresponding ligand is unknown. Ligands for RPTKsinclude mainly secreted small proteins, but also membrane-bound andextracellular matrix proteins.

[0522] Activation of RPTK by ligands involves ligand-mediated receptordimerization, resulting in transphosphorylation of the receptor subunitsand activation of the cytoplasmic tyrosine kinases. The cytoplasmictyrosine kinases include receptor associated tyrosine kinases of thesrc-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked andcytosolic protein tyrosine kinases, such as the Jak family, members ofwhich mediate signal transduction triggered by the cytokine superfamilyof receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).

[0523] Because of the wide range of known factors capable of stimulatingtyrosine kinase activity, identifying whether D-SLAM or a moleculeinduced by D-SLAM is capable of activating tyrosine kinase signaltransduction pathways is of interest. Therefore, the following protocolis designed to identify such molecules capable of activating thetyrosine kinase signal transduction pathways.

[0524] Seed target cells (e.g., primary keratinocytes) at a density ofapproximately 25,000 cells per well in a 96 well Loprodyne Silent ScreenPlates purchased from Nalge Nunc (Naperville, Ill.). The plates aresterilized with two 30 minute rinses with 100% ethanol, rinsed withwater and dried overnight. Some plates are coated for 2 hr with 100 mlof cell culture grade type I collagen (50 mg/ml), gelatin (2%) orpolylysine (50 mg/ml), all of which can be purchased from SigmaChemicals (St. Louis, Mo.) or 10% Matrigel purchased from BectonDickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at4 degree C. Cell growth on these plates is assayed by seeding 5,000cells/well in growth medium and indirect quantitation of cell numberthrough use of alamarBlue as described by the manufacturer AlamarBiosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers#3071 from Becton Dickinson (Bedford, Mass.) are used to cover theLoprodyne Silent Screen Plates. Falcon Microtest III cell culture platescan also be used in some proliferation experiments.

[0525] To prepare extracts, A431 cells are seeded onto the nylonmembranes of Loprodyne plates (20,000/200 ml/well) and culturedovernight in complete medium. Cells are quiesced by incubation inserum-free basal medium for 24 hr. After 5-20 minutes treatment with EGF(60 ng/ml) or 50 ul of the supernatant produced in Example 12, themedium was removed and 100 ml of extraction buffer ((20 mM HEPES pH 7.5,0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4, 2 mM Na4P2O7 and acocktail of protease inhibitors (# 1836170) obtained from BoeheringerMannheim (Indianapolis, Ind.) is added to each well and the plate isshaken on a rotating shaker for 5 minutes at 4° C. The plate is thenplaced in a vacuum transfer manifold and the extract filtered throughthe 0.45 mM membrane bottoms of each well using house vacuum. Extractsare collected in a 96-well catch/assay plate in the bottom of the vacuummanifold and immediately placed on ice. To obtain extracts clarified bycentrifugation, the content of each well, after detergent solubilizationfor 5 minutes, is removed and centrifuged for 15 minutes at 4 degree C.at 16,000×g.

[0526] Test the filtered extracts for levels of tyrosine kinaseactivity. Although many methods of detecting tyrosine kinase activityare known, one method is described here.

[0527] Generally, the tyrosine kinase activity of a supernatant isevaluated by determining its ability to phosphorylate a tyrosine residueon a specific substrate (a biotinylated peptide). Biotinylated peptidesthat can be used for this purpose include PSK1 (corresponding to aminoacids 6-20 of the cell division kinase cdc2-p34) and PSK2 (correspondingto amino acids 1-17 of gastrin). Both peptides are substrates for arange of tyrosine kinases and are available from Boehringer Mannheim.

[0528] The tyrosine kinase reaction is set up by adding the followingcomponents in order. First, add 10 ul of 5 uM Biotinylated Peptide, then10 ul ATP/Mg₂₊ (5 mM ATP/50 mM MgCl₂), then 10 ul of 5× Assay Buffer (40mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mMEGTA, 100 mM MgCl₂, 5 mM MnCl₂, 0.5 mg/ml BSA), then 5 ul of SodiumVanadate (1 mM), and then 5 ul of water. Mix the components gently andpreincubate the reaction mix at 30 degree C. for 2 min. Initial thereaction by adding 10 ul of the control enzyme or the filteredsupernatant.

[0529] The tyrosine kinase assay reaction is then terminated by adding10 ul of 120 mM EDTA and place the reactions on ice.

[0530] Tyrosine kinase activity is determined by transferring 50 ulaliquot of reaction mixture to a microtiter plate (MTP) module andincubating at 37 degree C. for 20 min. This allows the streptavadincoated 96 well plate to associate with the biotinylated peptide. Washthe MTP module with 300 ul/well of PBS four times. Next add 75 ul ofanti-phospotyrosine antibody conjugated to horse radishperoxidase(anti-P-Tyr-POD(0.5 u/ml)) to each well and incubate at 37degree C. for one hour. Wash the well as above.

[0531] Next add 100 ul of peroxidase substrate solution (BoehringerMannheim) and incubate at room temperature for at least 5 mins (up to 30min). Measure the absorbance of the sample at 405 nm by using ELISAreader. The level of bound peroxidase activity is quantitated using anELISA reader and reflects the level of tyrosine kinase activity.

Example 21 High-Throughput Screening Assay Identifying PhosphorylationActivity

[0532] As a potential alternative and/or compliment to the assay ofprotein tyrosine kinase activity described in Example 20, an assay whichdetects activation (phosphorylation) of major intracellular signaltransduction intermediates can also be used. For example, as describedbelow one particular assay can detect tyrosine phosphorylation of theErk-1 and Erk-2 kinases. However, phosphorylation of other molecules,such as Raf, JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src,Muscle specific kinase (MuSK), RAK, Tec, and Janus, as well as any otherphosphoserine, phosphotyrosine, or phosphothreonine molecule, can bedetected by substituting these molecules for Erk-1 or Erk-2 in thefollowing assay.

[0533] Specifically, assay plates are made by coating the wells of a96-well ELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at roomtemp, (RT). The plates are then rinsed with PBS and blocked with 3%BSA/PBS for 1 hr at RT. The protein G plates are then treated with 2commercial monoclonal antibodies (100 ng/well) against Erk-1 and Erk-2(1 hr at RT) (Santa Cruz Biotechnology). (To detect other molecules,this step can easily be modified by substituting a monoclonal antibodydetecting any of the above described molecules.) After 3-5 rinses withPBS, the plates are stored at 4 degree C. until use.

[0534] A431 cells are seeded at 20,000/well in a 96-well Loprodynefilterplate and cultured overnight in growth medium. The cells are thenstarved for 48 hr in basal medium (DMEM) and then treated with EGF (6ng/well) or 50 ul of the supernatants obtained in Example 12 for 5-20minutes. The cells are then solubilized and extracts filtered directlyinto the assay plate.

[0535] After incubation with the extract for 1 hr at RT, the wells areagain rinsed. As a positive control, a commercial preparation of MAPkinase (10 ng/well) is used in place of A431extract. Plates are thentreated with a commercial polyclonal (rabbit) antibody (1 ug/ml) whichspecifically recognizes the phosphorylated epitope of the Erk-1 andErk-2 kinases (1 hr at RT). This antibody is biotinylated by standardprocedures. The bound polyclonal antibody is then quantitated bysuccessive incubations with Europium-streptavidin and Europiumfluorescence enhancing reagent in the Wallac DELFIA instrument(time-resolved fluorescence). An increased fluorescent signal overbackground indicates a phosphorylation by D-SLAM or a molecule inducedby D-SLAM.

Example 22 Method of Determining Alterations in the D-SLAM Gene

[0536] RNA isolated from entire families or individual patientspresenting with a phenotype of interest (such as a disease) is beisolated. cDNA is then generated from these RNA samples using protocolsknown in the art. (See, Sambrook.) The cDNA is then used as a templatefor PCR, employing primers surrounding regions of interest in SEQ IDNO:1. Suggested PCR conditions consist of 35 cycles at 95 degree C. for30 seconds; 60-120 seconds at 52-58 degree C.; and 60-120 seconds at 70degree C., using buffer solutions described in Sidransky, D., et al.,Science 252:706 (1991).

[0537] PCR products are then sequenced using primers labeled at their 5′end with T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons ofD-SLAM is also determined and genomic PCR products analyzed to confirmthe results. PCR products harboring suspected mutations in D-SLAM isthen cloned and sequenced to validate the results of the directsequencing.

[0538] PCR products of D-SLAM are cloned into T-tailed vectors asdescribed in Holton, T. A. and Graham, M. W., Nucleic Acids Research,19:1156 (1991) and sequenced with T7 polymerase (United StatesBiochemical). Affected individuals are identified by mutations in D-SLAMnot present in unaffected individuals.

[0539] Genomic rearrangements are also observed as a method ofdetermining alterations in the D-SLAM gene. Genomic clones isolatedaccording to Example 2 are nick-translated with digoxigenindeoxy-uridine5′-triphosphate (Boehringer Manheim), and FISH performed as described inJohnson, Cg. et al., Methods Cell Biol. 35:73-99 (1991). Hybridizationwith the labeled probe is carried out using a vast excess of human cot-1DNA for specific hybridization to the D-SLAM genomic locus.

[0540] Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson, Cv. et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region of D-SLAM (hybridized by the probe)are identified as insertions, deletions, and translocations. TheseD-SLAM alterations are used as a diagnostic marker for an associateddisease.

Example 23 Method of Detecting Abnormal Levels of D-SLAM in a BiologicalSample

[0541] D-SLAM polypeptides can be detected in a biological sample, andif an increased or decreased level of D-SLAM is detected, thispolypeptide is a marker for a particular phenotype. Methods of detectionare numerous, and thus, it is understood that one skilled in the art canmodify the following assay to fit their particular needs.

[0542] For example, antibody-sandwich ELISAs are used to detect D-SLAMin a sample, preferably a biological sample. Wells of a microtiter plateare coated with specific antibodies to D-SLAM, at a final concentrationof 0.2 to 10 ug/ml. The antibodies are either monoclonal or polyclonaland are produced by the method described in Example 11. The wells areblocked so that non-specific binding of D-SLAM to the well is reduced.

[0543] The coated wells are then incubated for >2 hours at RT with asample containing D-SLAM. Preferably, serial dilutions of the sampleshould be used to validate results. The plates are then washed threetimes with deionized or distilled water to remove unbounded D-SLAM.

[0544] Next, 50 ul of specific antibody-alkaline phosphatase conjugate,at a concentration of 25-400 ng, is added and incubated for 2 hours atroom temperature. The plates are again washed three times with deionizedor distilled water to remove unbounded conjugate.

[0545] Add 75 ul of 4-methylumbelliferyl phosphate (MUP) orp-nitrophenyl phosphate (NPP) substrate solution to each well andincubate 1 hour at room temperature. Measure the reaction by amicrotiter plate reader. Prepare a standard curve, using serialdilutions of a control sample, and plot D-SLAM polypeptide concentrationon the X-axis (log scale) and fluorescence or absorbance of the Y-axis(linear scale). Interpolate the concentration of the D-SLAM in thesample using the standard curve.

Example 24 Formulating a Polypeptide

[0546] The D-SLAM composition will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient (especially the side effects oftreatment with the D-SLAM polypeptide alone), the site of delivery, themethod of administration, the scheduling of administration, and otherfactors known to practitioners. The “effective amount” for purposesherein is thus determined by such considerations.

[0547] As a general proposition, the total pharmaceutically effectiveamount of D-SLAM administered parenterally per dose will be in the rangeof about 1 ug/kg/day to 10 mg/kg/day of patient body weight, although,as noted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day, and most preferablyfor humans between about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, D-SLAM is typically administered at a dose rate of about 1ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injections per day orby continuous subcutaneous infusions, for example, using a mini-pump. Anintravenous bag solution may also be employed. The length of treatmentneeded to observe changes and the interval following treatment forresponses to occur appears to vary depending on the desired effect.

[0548] Pharmaceutical compositions containing D-SLAM are administeredorally, rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, gels, drops ortransdermal patch), bucally, or as an oral or nasal spray.“Pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrastemal, subcutaneous andintraarticular injection and infusion.

[0549] D-SLAM is also suitably administered by sustained-releasesystems. Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556(1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J.Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech.12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include liposomally entrapped D-SLAM polypeptides.Liposomes containing the D-SLAM are prepared by methods known per se: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034 (1980); EP52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat.Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal secreted polypeptide therapy.

[0550] For parenteral administration, in one embodiment, D-SLAM isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

[0551] Generally, the formulations are prepared by contacting D-SLAMuniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

[0552] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[0553] D-SLAM is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

[0554] D-SLAM used for therapeutic administration can be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticpolypeptide compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

[0555] D-SLAM polypeptides ordinarily will be stored in unit ormulti-dose containers, for example, sealed ampoules or vials, as anaqueous solution or as a lyophilized formulation for reconstitution. Asan example of a lyophilized formulation, 10-ml vials are filled with 5ml of sterile-filtered 1% (w/v) aqueous D-SLAM polypeptide solution, andthe resulting mixture is lyophilized. The infusion solution is preparedby reconstituting the lyophilized D-SLAM polypeptide usingbacteriostatic Water-for-Injection.

[0556] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, D-SLAM may be employed in conjunction with other therapeuticcompounds.

[0557] The compositions of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the compositions of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

[0558] In one embodiment, the compositions of the invention areadministered in combination with other members of the TNF family. TNF,TNF-related or TNF-like molecules that may be administered with thecompositions of the invention include, but are not limited to, solubleforms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known asTNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL,FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (InternationalPublication No. WO 96/14328), AIM-I (International Publication No. WO97/33899), endokine-alpha (International Publication No. WO 98/07880),TR6 (International Publication No. WO 98/30694), OPG, andneutrokine-alpha (International Publication No. WO 98/18921, OX40, andnerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3(International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TR6 (International Publication No. WO 98/30694), TR7(International Publication No. WO 98/41629), TRANK, TR9 (InternationalPublication No. WO 98/56892), TR10 (International Publication No. WO98/54202), 312C2 (International Publication No. WO 98/06842), and TR12,and soluble forms CD154, CD70, and CD153.

[0559] Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

[0560] In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, tetracycline, metronidazole, amoxicillin,beta-lactamases, aminoglycosides, macrolides, quinolones,fluoroquinolones, cephalosporins, erythromycin, ciprofloxacin, andstreptomycin.

[0561] In an additional embodiment, the compositions of the inventionare administered alone or in combination with an anti-inflammatoryagent. Anti-inflammatory agents that may be administered with thecompositions of the invention include, but are not limited to,glucocorticoids and the nonsteroidal anti-inflammatories,aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acidderivatives, pyrazoles, pyrazolones, salicylic acid derivatives,thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone,nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime,proquazone, proxazole, and tenidap.

[0562] In another embodiment, compostions of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the compositionsof the invention include, but are not limited to, antibiotic derivatives(e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin);antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil,5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid,plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g.,carmustine, BCNU, lomustine, CCNU, cytosine arabinoside,cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin,busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0563] In an additional embodiment, the compositions of the inventionare administered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha.

[0564] In an additional embodiment, the compositions of the inventionare administered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the compositions of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PlGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PlGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B-186 (VEGF-B 186), as disclosed in InternationalPublication Number WO 96/26736; Vascular Endothelial Growth Factor-D(VEGF-D), as disclosed in International Publication Number WO 98/02543;Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/07832; and Vascular EndothelialGrowth Factor-E (VEGF-E), as disclosed in German Patent NumberDE19639601. The above mentioned references are incorporated herein byreference herein.

[0565] In an additional embodiment, the compositions of the inventionare administered in combination with Fibroblast Growth Factors.Fibroblast Growth Factors that may be administered with the compositionsof the invention include, but are not limited to, FGF-1, FGF-2, FGF-3,FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

[0566] In additional embodiments, the compositions of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Example 25 Method of Treating Decreased Levels of D-SLAM

[0567] The present invention relates to a method for treating anindividual in need of a decreased level of D-SLAM activity in the bodycomprising, administering to such an individual a composition comprisinga therapeutically effective amount of D-SLAM antagonist. Preferredantagonists for use in the present invention are D-SLAM-specificantibodies.

[0568] Moreover, it will be appreciated that conditions caused by adecrease in the standard or normal expression level of D-SLAM in anindividual can be treated by administering D-SLAM, preferably in thesecreted form. Thus, the invention also provides a method of treatmentof an individual in need of an increased level of D-SLAM polypeptidecomprising administering to such an individual a pharmaceuticalcomposition comprising an amount of D-SLAM to increase the activitylevel of D-SLAM in such an individual.

[0569] For example, a patient with decreased levels of D-SLAMpolypeptide receives a daily dose 0.1-100 ug/kg of the polypeptide forsix consecutive days. Preferably, the polypeptide is in the secretedform. The exact details of the dosing scheme, based on administrationand formulation, are provided in Example 24.

Example 26 Method of Treating Increased Levels of D-SLAM

[0570] The present invention also relates to a method for treating anindividual in need of an increased level of D-SLAM activity in the bodycomprising administering to such an individual a composition comprisinga therapeutically effective amount of D-SLAM or an agonist thereof.

[0571] Antisense technology is used to inhibit production of D-SLAM.This technology is one example of a method of decreasing levels ofD-SLAM polypeptide, preferably a secreted form, due to a variety ofetiologies, such as cancer.

[0572] For example, a patient diagnosed with abnormally increased levelsof D-SLAM is administered intravenously antisense polynucleotides at0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment isrepeated after a 7-day rest period if the treatment was well tolerated.The formulation of the antisense polynucleotide is provided in Example24.

Example 27 Method of Treatment Using Gene Therapy—Ex Vivo

[0573] One method of gene therapy transplants fibroblasts, which arecapable of expressing D-SLAM polypeptides, onto a patient. Generally,fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin) is added. The flasks are then incubated at 37 degreeC. for approximately one week.

[0574] At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

[0575] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flankedby the long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0576] The cDNA encoding D-SLAM can be amplified using PCR primers whichcorrespond to the 5′ and 3′ end sequences respectively as set forth inExample 1. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector containsproperly inserted D-SLAM.

[0577] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the D-SLAM gene is then added to the media and thepackaging cells transduced with the vector. The packaging cells nowproduce infectious viral particles containing the D-SLAM gene(thepackaging cells are now referred to as producer cells).

[0578] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his. Once thefibroblasts have been efficiently infected, the fibroblasts are analyzedto determine whether D-SLAM protein is produced.

[0579] The engineered fibroblasts are then transplanted onto the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads.

Example 28 Gene Therapy Using Endogenous D-SLAM Gene

[0580] Another method of gene therapy according to the present inventioninvolves operably associating the endogenous D-SLAM sequence with apromoter via homologous recombination as described, for example, in U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No.WO 96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989).This method involves the activation of a gene which is present in thetarget cells, but which is not expressed in the cells, or is expressedat a lower level than desired.

[0581] Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous D-SLAM, flanking the promoter. The targeting sequence willbe sufficiently near the 5′ end of D-SLAM so the promoter will beoperably linked to the endogenous sequence upon homologousrecombination. The promoter and the targeting sequences can be amplifiedusing PCR. Preferably, the amplified promoter contains distinctrestriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ endof the first targeting sequence contains the same restriction enzymesite as the 5′ end of the amplified promoter and the 5′ end of thesecond targeting sequence contains the same restriction site as the 3′end of the amplified promoter.

[0582] The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

[0583] In this Example, the polynucleotide constructs are administeredas naked polynucleotides via electroporation. However, thepolynucleotide constructs may also be administered withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, precipitating agents, etc. Such methods of delivery areknown in the art.

[0584] Once the cells are transfected, homologous recombination willtake place which results in the promoter being operably linked to theendogenous D-SLAM sequence. This results in the expression of D-SLAM inthe cell. Expression may be detected by immunological staining, or anyother method known in the art.

[0585] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in DMEM + 10% fetal calf serum. Exponentiallygrowing or early stationary phase fibroblasts are trypsinized and rinsedfrom the plastic surface with nutrient medium. An aliquot of the cellsuspension is removed for counting, and the remaining cells aresubjected to centrifugation. The supernatant is aspirated and the pelletis resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3,137 mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

[0586] Plasmid DNA is prepared according to standard techniques. Forexample, to construct a plasmid for targeting to the D-SLAM locus,plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII.The CMV promoter is amplified by PCR with an XbaI site on the 5′ end anda BamHI site on the 3′end. Two D-SLAM non-coding sequences are amplifiedvia PCR: one D-SLAM non-coding sequence (D-SLAM fragment 1) is amplifiedwith a HindIII site at the 5′ end and an Xba site at the 3′end; theother D-SLAM non-coding sequence (D-SLAM fragment 2) is amplified with aBamHI site at the 5′end and a HindIII site at the 3′end. The CMVpromoter and D-SLAM fragments are digested with the appropriate enzymes(CMV promoter—XbaI and BamHI; D-SLAM fragment 1—XbaI; D-SLAM fragment2—BamHI) and ligated together. The resulting ligation product isdigested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

[0587] Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrodegap (Bio-Rad). The final DNA concentration is generally at least 120μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5×10⁶cells) is then added to the cuvette, and the cell suspension and DNAsolutions are gently mixed. Electroporation is performed with aGene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960μF and 250-300 V, respectively. As voltage increases, cell survivaldecreases, but the percentage of surviving cells that stably incorporatethe introduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

[0588] Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hours.

[0589] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product. Thefibroblasts can then be introduced into a patient as described above.

Example 29 Method of Treatment Using Gene Therapy—In Vivo

[0590] Another aspect of the present invention is using in vivo genetherapy methods to treat disorders, diseases and conditions. The genetherapy method relates to the introduction of naked nucleic acid (DNA,RNA, and antisense DNA or RNA) D-SLAM sequences into an animal toincrease or decrease the expression of the D-SLAM polypeptide. TheD-SLAM polynucleotide may be operatively linked to a promoter or anyother genetic elements necessary for the expression of the D-SLAMpolypeptide by the target tissue. Such gene therapy and deliverytechniques and methods are known in the art, see, for example, WO90/11092, WO 98/11779; U.S. Pat. Nos. 5,693,622, 5,705,151, 5,580,859;Tabata H. et al. (1997) Cardiovasc. Res. 35(3):470-479, Chao J et al.(1997) Pharmacol. Res. 35(6):517-522, Wolff J. A. (1997) Neuromuscul.Disord. 7(5):314-318, Schwartz B. et al. (1996) Gene Ther. 3(5):405-411,Tsurumi Y. et al. (1996) Circulation 94(12):3281-3290 (incorporatedherein by reference).

[0591] The D-SLAM polynucleotide constructs may be delivered by anymethod that delivers injectable materials to the cells of an animal,such as, injection into the interstitial space of tissues (heart,muscle, skin, lung, liver, intestine and the like). The D-SLAMpolynucleotide constructs can be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

[0592] The term “naked” polynucleotide, DNA or RNA, refers to sequencesthat are free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the D-SLAM polynucleotides may also be delivered inliposome formulations (such as those taught in Felgner P. L. et al.(1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995)Biol. Cell 85(1):1-7) which can be prepared by methods well known tothose skilled in the art.

[0593] The D-SLAM polynucleotide vector constructs used in the genetherapy method are preferably constructs that will not integrate intothe host genome nor will they contain sequences that allow forreplication. Any strong promoter known to those skilled in the art canbe used for driving the expression of DNA. Unlike other gene therapiestechniques, one major advantage of introducing naked nucleic acidsequences into target cells is the transitory nature of thepolynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

[0594] The D-SLAM polynucleotide construct can be delivered to theinterstitial space of tissues within the an animal, including of muscle,skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

[0595] For the naked D-SLAM polynucleotide injection, an effectivedosage amount of DNA or RNA will be in the range of from about 0.05 g/kgbody weight to about 50 mg/kg body weight. Preferably the dosage will befrom about 0.005 mg/kg to about 20 mg/kg and more preferably from about0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skillwill appreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked D-SLAMpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

[0596] The dose response effects of injected D-SLAM polynucleotide inmuscle in vivo is determined as follows. Suitable D-SLAM template DNAfor production of mRNA coding for D-SLAM polypeptide is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

[0597] Five to six week old female and male Balb/C mice are anesthetizedby intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cmincision is made on the anterior thigh, and the quadriceps muscle isdirectly visualized. The D-SLAM template DNA is injected in 0.1 ml ofcarrier in a 1 cc syringe through a 27 gauge needle over one minute,approximately 0.5 cm from the distal insertion site of the muscle intothe knee and about 0.2 cm deep. A suture is placed over the injectionsite for future localization, and the skin is closed with stainlesssteel clips.

[0598] After an appropriate incubation time (e.g., 7 days) muscleextracts are prepared by excising the entire quadriceps. Every fifth 15um cross-section of the individual quadriceps muscles is histochemicallystained for D-SLAM protein expression. A time course for D-SLAM proteinexpression may be done in a similar fashion except that quadriceps fromdifferent mice are harvested at different times. Persistence of D-SLAMDNA in muscle following injection may be determined by Southern blotanalysis after preparing total cellular DNA and HIRT supernatants frominjected and control mice. The results of the above experimentation inmice can be use to extrapolate proper dosages and other treatmentparameters in humans and other animals using D-SLAM naked DNA.

Example 30 D-SLAM Transgenic Animals

[0599] The D-SLAM polypeptides can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep,cows and non-human primates, e.g., baboons, monkeys, and chimpanzees maybe used to generate transgenic animals. In a specific embodiment,techniques described herein or otherwise known in the art, are used toexpress polypeptides of the invention in humans, as part of a genetherapy protocol.

[0600] Any technique known in the art may be used to introduce thetransgene (i.e., polynucleotides of the invention) into animals toproduce the founder lines of transgenic animals. Such techniquesinclude, but are not limited to, pronuclear microinjection (Paterson etal., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al.,Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology(NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191(1989)); retrovirus mediated gene transfer into germ lines (Van derPutten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)),blastocysts or embryos; gene targeting in embryonic stem cells (Thompsonet al., Cell 56:313-321 (1989)); electroporation of cells or embryos(Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction ofthepolynucleotides of the invention using a gene gun (see, e.g., Ulmeret al., Science 259:1745 (1993); introducing nucleic acid constructsinto embryonic pleuripotent stem cells and transferring the stem cellsback into the blastocyst; and sperm-mediated gene transfer (Lavitrano etal., Cell 57:717-723 (1989); etc. For a review of such techniques, seeGordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989),which is incorporated by reference herein in its entirety.

[0601] Any technique known in the art may be used to produce transgenicclones containing polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

[0602] The present invention provides for transgenic animals that carrythe transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.

[0603] Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous geneare designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous gene. Thetransgene may also be selectively introduced into a particular celltype, thus inactivating the endogenous gene in only that cell type, byfollowing, for example, the teaching of Gu et al. (Gu et al., Science265:103-106 (1994)). The regulatory sequences required for such acell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art. Thecontents of each of the documents recited in this paragraph is hereinincorporated by reference in its entirety.

[0604] Any of the D-SLAM polypeptides disclose throughout thisapplication can be used to generate transgenic animals. For example, DNAencoding amino acids M1-K232 of SEQ ID NO:2 can be inserted into avector containing a promoter, such as the actin promoter, which willubiquitously express the inserted fragment. Primers that can be used togenerate such fragments include a 5′ primer containing a BamHIrestriction site shown in bold:

[0605] GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCT C (SEQ ID NO:22) and a 3′ primer, containing a Xba restriction site shown in bold:

[0606] GCAGCATCTAGATTATTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO: 24).This construct will express the predicted extracellular domain of D-SLAMunder the control of the actin promoter for ubiquitous expression. Theregion of D-SLAM included in this construct extends from M1-K232 of SEQID NO:2.

[0607] Similarly, the DNA encoding the full length D-SLAM protein canalso be inserted into a vector using the following primers: A 5′ primercontaining a BamHI restriction site shown in bold:

[0608] GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCT C (SEQ ID NO:22) and a 3′ primer, containing a Xba restriction site shown in bold:

[0609] GCAGCATCTAGATTATGGCAGATCCTGCACAAGGGGGTTCTCTGTC (SEQ ID NO: 23).Besides these two examples, other fragments of D-SLAM can also beinserted into a vector to create transgenics having ubiquitousexpression.

[0610] Alternatively, polynucleotides of the invention can be insertedin a vector which controls tissue specific expression through a tissuespecific promoter. For example, a construct having a transferrinpromoter would express the D-SLAM polypeptide in the liver of transgenicanimals. Therefore, DNA encoding amino acids M1-K232 of SEQ ID NO:2 canbe amplified using a 5′ primer, having a BamHI restriction site shown inbold:

[0611] GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCT C (SEQ ID NO:22), and a 3′ primer, containing a Xba restriction site shown in bold:

[0612] GCAGCATCTAGATTATTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO: 24).

[0613] Similarly, the DNA encoding the full length D-SLAM protein canalso be inserted into a vector for tissue specific expression using thefollowing primers: A 5′ primer containing a BamHI restriction site shownin bold:

[0614] GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCT C (SEQ ID NO:22) and a 3′ primer, containing a Xba restriction site shown in bold:

[0615] GCAGCATCTAGATTATGGCAGATCCTGCACAAGGGGGTTCTCTGTC (SEQ ID NO: 23).

[0616] Once transgenic animals have been generated, the expression ofthe recombinant gene may be assayed utilizing standard techniques.Initial screening may. be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

[0617] Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

[0618] Transgenic animals of the invention have uses which include, butare not limited to, animal model systems useful in elaborating thebiological function of D-SLAM polypeptides, studying conditions and/ordisorders associated with aberrant D-SLAM expression, and in screeningfor compounds effective in ameliorating such conditions and/ordisorders.

Example 31 D-SLAM Knock-Out Animals

[0619] Endogenous D-SLAM gene expression can also be reduced byinactivating or “knocking out” the D-SLAM gene and/or its promoter usingtargeted homologous recombination. (E.g., see Smithies et al., Nature317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompsonet al., Cell 5:313-321 (1989); each of which is incorporated byreference herein in its entirety). For example, a mutant, non-functionalpolynucleotide of the invention (or a completely unrelated DNA sequence)flanked by DNA homologous to the endogenous polynucleotide sequence(either the coding regions or regulatory regions of the gene) can beused, with or without a selectable marker and/or a negative selectablemarker, to transfect cells that express polypeptides of the invention invivo. In another embodiment, techniques known in the art are used togenerate knockouts in cells that contain, but do not express the gene ofinterest. Insertion of the DNA construct, via targeted homologousrecombination, results in inactivation of the targeted gene. Suchapproaches are particularly suited in research and agricultural fieldswhere modifications to embryonic stem cells can be used to generateanimal offspring with an inactive targeted gene (e.g., see Thomas &Capecchi 1987 and Thompson 1989, supra). However this approach can beroutinely adapted for use in humans provided the recombinant DNAconstructs are directly administered or targeted to the required site invivo using appropriate viral vectors that will be apparent to those ofskill in the art.

[0620] In further embodiments of the invention, cells that aregenetically engineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe D-SLAM polypeptides. The engineered cells which express andpreferably secrete the polypeptides of the invention can be introducedinto the patient systemically, e.g., in the circulation, orintraperitoneally.

[0621] Alternatively, the cells can be incorporated into a matrix andimplanted in the body, e.g., genetically engineered fibroblasts can beimplanted as part of a skin graft; genetically engineered endothelialcells can be implanted as part of a lymphatic or vascular graft. (See,for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan &Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated byreference herein in its entirety).

[0622] When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

[0623] Knock-out animals of the invention have uses which include, butare not limited to, animal model systems useful in elaborating thebiological function of D-SLAM polypeptides, studying conditions and/ordisorders associated with aberrant D-SLAM expression, and in screeningfor compounds effective in ameliorating such conditions and/ordisorders.

Example 32 Assays Detecting Stimulation or Inhibition of B CellProliferation and Differentiation

[0624] Generation of functional humoral immune responses requires bothsoluble and cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, L-4, IL-5,IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signalsare by themselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.

[0625] One of the best studied classes of B-cell co-stimulatory proteinsis the TNF-superfamily. Within this family CD40, CD27, and CD30 alongwith their respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

[0626] In Vitro Assay

[0627] Purified D-SLAM protein, or truncated forms thereof, is assessedfor its ability to induce activation, proliferation, differentiation orinhibition and/or death in B-cell populations and their precursors. Theactivity of D-SLAM protein on purified human tonsillar B cells, measuredqualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessedin a standard B-lymphocyte co-stimulation assay in which purifiedtonsillar B cells are cultured in the presence of either formalin-fixedStaphylococcus aureus Cowan I (SAC) or immobilized anti-human IgMantibody as the priming agent. Second signals such as IL-2 and IL-15synergize with SAC and IgM crosslinking to elicit B cell proliferationas measured by tritiated-thymidine incorporation. Novel synergizingagents can be readily identified using this assay. The assay involvesisolating human tonsillar B cells by magnetic bead (MACS) depletion ofCD3-positive cells. The resulting cell population is greater than 95% Bcells as assessed by expression of CD45R(B220).

[0628] Various dilutions of each sample are placed into individual wellsof a 96-well plate to which are added 10⁵ B-cells suspended in culturemedium (RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100U/ml penicillin,10 ug/ml streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of150 ul. Proliferation or inhibition is quantitated by a 20 h pulse (1uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

[0629] In Vivo Assay

[0630] BALB/c mice are injected (i.p.) twice per day with buffer only,or 2 mg/Kg of D-SLAM protein, or truncated forms thereof. Mice receivethis treatment for 4 consecutive days, at which time they are sacrificedand various tissues and serum collected for analyses. Comparison of H&Esections from normal and D-SLAM protein-treated spleens identify theresults of the activity of D-SLAM protein on spleen cells, such as thediffusion of peri-arterial lymphatic sheaths, and/or significantincreases in the nucleated cellularity of the red pulp regions, whichmay indicate the activation of the differentiation and proliferation ofB-cell populations. Immunohistochemical studies using a B cell marker,anti-CD45R(B220), are used to determine whether any physiologicalchanges to splenic cells, such as splenic disorganization, are due toincreased B-cell representation within loosely defined B-cell zones thatinfiltrate established T-cell regions.

[0631] Flow cytometric analyses of the spleens from D-SLAMprotein-treated mice is used to indicate whether D-SLAM proteinspecifically increases the proportion of ThB+, CD45R(B220)dull B cellsover that which is observed in control mice.

[0632] Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andD-SLAM protein-treated mice.

[0633] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 33 T Cell Proliferation Assay

[0634] A CD3-induced proliferation assay is performed on PBMCs and ismeasured by the uptake of ³H-thymidine. The assay is performed asfollows. Ninety-six well plates are coated with 100 μl/well of mAb toCD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33.1) overnightat 4 degree C. (1 μg/ml in 0.05M bicarbonate buffer, pH 9.5), thenwashed three times with PBS. PBMC are isolated by F/H gradientcentrifugation from human peripheral blood and added to quadruplicatewells (5×10⁴/well) of mAb coated plates in RPMI containing 10% FCS andP/S in the presence of varying concentrations of D-SLAM protein (totalvolume 200 μl). Relevant protein buffer and medium alone are controls.After 48 hr. culture at 37 degree C., plates are spun for 2 min. at 1000rpm and 100 μl of supernatant is removed and stored −20 degree C. formeasurement of IL-2 (or other cytokines) if effect on proliferation isobserved. Wells are supplemented with 100 μl of medium containing 0.5μCi of ³H-thymidine and cultured at 37 degree C. for 18-24 hr. Wells areharvested and incorporation of ³H-thymidine used as a measure ofproliferation. Anti-CD3 alone is the positive control for proliferation.IL-2 (100U/ml) is also used as a control which enhances proliferation.Control antibody which does not induce proliferation of T cells is usedas the negative controls for the effects of D-SLAM proteins.

[0635] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 34 Effect of D-SLAM on the Expression of MHC Class II,Costimulatory and Adhesion Molecules and Cell Differentiation ofMonocytes and Monocyte-Derived Human Dendritic Cells

[0636] Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-α,causes a rapid change in surface phenotype (increased expression of MHCclass I and II, costimulatory and adhesion molecules, downregulation ofFCγRII, upregulation of CD83). These changes correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

[0637] FACS analysis of surface antigens is performed as follows. Cellsare treated 1-3 days with increasing concentrations of D-SLAM or LPS(positive control), washed with PBS containing 1% BSA and 0.02 mM sodiumazide, and then incubated with 1:20 dilution of appropriate FITC- orPE-labeled monoclonal antibodies for 30 minutes at 4° C. After anadditional wash, the labeled cells are analyzed by flow cytometry on aFACScan (Becton Dickinson).

[0638] Effect on the Production of Cytokines

[0639] Cytokines generated by dendritic cells, in particular IL-12, areimportant in the initiation of T-cell dependent immune responses. IL-12strongly influences the development of Th1 helper T-cell immuneresponse, and induces cytotoxic T and NK cell function. An ELISA is usedto measure the IL-12 release as follows. Dendritic cells (10⁶/ml) aretreated with increasing concentrations of D-SLAM for 24 hours. LPS (100ng/ml) is added to the cell culture as positive control. Supernatantsfrom the cell cultures are then collected and analyzed for IL-12 contentusing commercial ELISA kit (e..g, R & D Systems (Minneapolis, Minn.)).The standard protocols provided with the kits are used.

[0640] Effect on the Expression of MHC Class II, Costimulatory andAdhesion Molecules

[0641] Three major families of cell surface antigens can be identifiedon monocytes: adhesion molecules, molecules involved in antigenpresentation, and Fc receptor. Modulation of the expression of MHC classII antigens and other costimulatory molecules, such as B7 and ICAM-1,may result in changes in the antigen presenting capacity of monocytesand ability to induce T cell activation. Increase expression of Fcreceptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

[0642] FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations of D-SLAMor LPS (positive control), washed with PBS containing 1% BSA and 0.02 mMsodium azide, and then incubated with 1:20 dilution of appropriate FITC-or PE-labeled monoclonal antibodies for 30 minutes at 4° C. After anadditional wash, the labeled cells are analyzed by flow cytometry on aFACScan (Becton Dickinson).

[0643] Monocyte Activation and/or Increased Survival

[0644] Assays for molecules that activate (or alternatively, inactivate)monocytes and/or increase monocyte survival (or alternatively, decreasemonocyte survival) are known in the art and may routinely be applied todetermine whether a molecule of the invention functions as an inhibitoror activator of monocytes. D-SLAM, agonists, or antagonists of D-SLAMcan be screened using the three assays described below. For each ofthese assays, Peripheral blood mononuclear cells (PBMC) are purifiedfrom single donor leukopacks (American Red Cross, Baltimore, Md.) bycentrifugation through a Histopaque gradient (Sigma). Monocytes areisolated from PBMC by counterflow centrifugal elutriation.

[0645] Monocyte Survival Assay

[0646] Human peripheral blood monocytes progressively lose viabilitywhen cultured in absence of serum or other stimuli. Their death resultsfrom internally regulated process (apoptosis). Addition to the cultureof activating factors, such as TNF-alpha dramatically improves cellsurvival and prevents DNA fragmentation. Propidium iodide (PI) stainingis used to measure apoptosis as follows. Monocytes are cultured for 48hours in polypropylene tubes in serum-free medium (positive control), inthe presence of 100 ng/ml TNF-alpha (negative control), and in thepresence of varying concentrations of the compound to be tested. Cellsare suspended at a concentration of 2×10⁶/ml in PBS containing PI at afinal concentration of 5 μg/ml, and then incubaed at room temperaturefor 5 minutes before FACScan analysis. PI uptake has been demonstratedto correlate with DNA fragmentation in this experimental paradigm.

[0647] Effect on Cytokine Release

[0648] An important function of monocytes/macrophages is theirregulatory activity on other cellular populations of the immune systemthrough the release of cytokines after stimulation. An ELISA to measurecytokine release is performed as follows. Human monocytes are incubatedat a density of 5×10⁵ cells/ml with increasing concentrations of D-SLAMand under the same conditions, but in the absence of D-SLAM. For IL-12production, the cells are primed overnight with TFN (100 U/ml) inpresence of D-SLAM. LPS (10 ng/ml) is then added. Conditioned media arecollected after 24 h and kept frozen until use. Measurement ofTNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a commerciallyavailable ELISA kit (e..g, R & D Systems (Minneapolis, Minn.)) andapplying the standard protocols provided with the kit.

[0649] Oxidative Burst

[0650] Purified monocytes are plated in 96-w plate at 2×10⁵ cell/well.Increasing concentrations of D-SLAM are added to the wells in a totalvolume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamine andantibiotics). After 3 days incubation, the plates are centrifuged andthe medium is removed from the wells. To the macrophage monolayers, 0.2ml per well of phenol red solution (140 mM NaCl, 10 mM potassiumphosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/mlof HRPO) is added, together with the stimulant (200 nM PMA). The platesare incubated at 37° C. for 2 hours and the reaction is stopped byadding 20 μl 1N NaOH per well. The absorbance is read at 610 nm. Tocalculate the amount of H₂O₂ produced by the macrophages, a standardcurve of a H₂O₂ solution of known molarity is performed for eachexperiment.

[0651] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

[0652] Results

[0653] As shown below, D-SLAM appears to specifically bindmonocyte-derived dendritic cells, while in this experiment, D-SLAM didnot bind to monocytes, T cells, or a variety of cell lines. Meanfluorescence Control D-SLAM Dendritic cells 3.94 10.95 Monocytes 3.183.18 T cells 10.84 10.85 @ CD3 activ. T cells 5.94 5.93 IM-9 4.1 4.51K562 2.65 2.89 SupT13 2.63 3.76 Jurkat 3.37 3.62 THP-1 3.92 4.71

[0654] Moreover, following incubation of dendritic cells with D-SLAM, anupregulation of a number of cell surface markers that are indicative ofdendritic cell maturation or activation are observed (see below). Thus,D-SLAM may be involved in dendritic cell activation and maturation. Meanfluorescence Controls HLA-DR CD54 CD86 CD83 Untreated 6/5 1597 325  23 4 D-SLAM 10 6/4 2432 488  43  5 D-SLAM 100 5/3 3545 309 154 10 D-SLAM1000 8/3 2952 657 258 18

[0655] Additional data suggests that D-SLAM may induce TNF-alphaproduction by monocytes, and that D-SLAM may bind to itself in ahomotypic association. Therefore, D-SLAM would likely activate any cell,including non-hematopoietic cells, such as stromal cells, having D-SLAMexpressed on the cell surface.

[0656] Thus, activating/maturing dendritic cells by increasing theamount of D-SLAM in a patient could be useful in the treatment of cancerand modulating the immune response. For example, if increasing theamount of D-SLAM elevates cell surface expression of dendritic cellmarkers, such as CD83, immune responses against tumors may be activated.In such an instance, treatment of patients having tumors with a solubleversion of D-SLAM could lead to upregulation of CD83 on dendritic cellsand result in enhanced recognition of tumors leading to an enhancedrejection/killing of the tumor.

Example 35 D-SLAM Biological Effects

[0657] Astrocyte and Neuronal Assays*

[0658] Recombinant D-SLAM, expressed and purified as described above,can be tested for activity in promoting the survival, neurite outgrowth,or phenotypic differentiation of cortical neuronal cells and forinducing the proliferation of glial fibrillary acidic proteinimmunopositive cells, astrocytes. The selection of cortical cells forthe bioassay is based on the prevalent expression of FGF-1 and FGF-2 incortical structures and on the previously reported enhancement ofcortical neuronal survival resulting from FGF-2 treatment. A thymidineincorporation assay, for example, can be used to elucidate D-SLAM'sactivity on these cells.

[0659] Moreover, previous reports describing the biological effects ofFGF-2 (basic FGF) on cortical or hippocampal neurons in vitro havedemonstrated increases in both neuron survival and neurite outgrowth(Walicke, P. et al., “Fibroblast growth factor promotes survival ofdissociated hippocampal neurons and enhances neurite extension.” Proc.Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated byreference in its entirety). However, reports from experiments done onPC-12 cells suggest that these two responses are not necessarilysynonymous and may depend on not only which FGF is being tested but alsoon which receptor(s) are expressed on the target cells. Using theprimary cortical neuronal culture paradigm, the ability of D-SLAM toinduce neurite outgrowth can be compared to the response achieved withFGF-2 using, for example, a thyrnidine incorporation assay.

[0660] Fibroblast and Endothelial Cell Assays*

[0661] Human lung fibroblasts are obtained from Clonetics (San Diego,Calif.) and maintained in growth media from Clonetics. Dermalmicrovascular endothelial cells are obtained from Cell Applications (SanDiego, Calif.). For proliferation assays, the human lung fibroblasts anddermal microvascular endothelial cells can be cultured at 5,000cells/well in a 96-well plate for one day in growth medium. The cellsare then incubated for one day in 0.1% BSA basal medium. After replacingthe medium with fresh 0.1% BSA medium, the cells are incubated with thetest proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento,Calif.) is added to each well to a final concentration of 10%. The cellsare incubated for 4 hr. Cell viability is measured by reading in aCytoFluor fluorescence reader. For the PGE₂ assays, the human lungfibroblasts are cultured at 5,000 cells/well in a 96-well plate for oneday. After a medium change to 0.1% BSA basal medium, the cells areincubated with FGF-2 or D-SLAM with or without IL-1α for 24 hours. Thesupernatants are collected and assayed for PGE₂ by EIA kit (Cayman, AnnArbor, Mich.). For the IL-6 assays, the human lung fibroblasts arecultured at 5,000 cells/well in a 96-well plate for one day. After amedium change to 0.1% BSA basal medium, the cells are incubated withFGF-2 or D-SLAM with or without IL-1α for 24 hours. The supernatants arecollected and assayed for IL-6 by ELISA kit (Endogen, Cambridge, Mass.).

[0662] Human lung fibroblasts are cultured with FGF-2 or D-SLAM for 3days in basal medium before the addition of Alamar Blue to assesseffects on growth of the fibroblasts. FGF-2 should show a stimulation at10-2500 ng/ml which can be used to compare stimulation with D-SLAM.

[0663] Parkinson Models

[0664] The loss of motor function in Parkinson's disease is attributedto a deficiency of striatal dopamine resulting from the degeneration ofthe nigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine(MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP⁺) and released.Subsequently, MPP⁺ is actively accumulated in dopaminergic neurons bythe high-affinity reuptake transporter for dopamine. MPP⁺ is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

[0665] It has been demonstrated in tissue culture paradigms that FGF-2(basic FGF) has trophic activity towards nigral dopaminergic neurons(Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

[0666] Based on the data with FGF-2, D-SLAM can be evaluated todetermine whether it has an action similar to that of FGF-2 in enhancingdopaminergic neuronal survival in vitro and it can also be tested invivo for protection of dopaminergic neurons in the striatum from thedamage associated with MPTP treatment. The potential effect of D-SLAM isfirst examined in vitro in a dopaminergic neuronal cell cultureparadigm. The cultures are prepared by dissecting the midbrain floorplate from gestation day 14 Wistar rat embryos. The tissue isdissociated with trypsin and seeded at a density of 200,000 cells/cm² onpolyorthinine-laminin coated glass coverslips. The cells are maintainedin Dulbecco's Modified Eagle's medium and F12 medium containing hormonalsupplements (N1). The cultures are fixed with paraformaldehyde after 8days in vitro and are processed for tyrosine hydroxylase, a specificmarker for dopminergic neurons, immunohistochemical staining.Dissociated cell cultures are prepared from embryonic rats. The culturemedium is changed every third day and the factors are also added at thattime.

[0667] Since the dopaminergic neurons are isolated from animals atgestation day 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, an increase in thenumber of tyrosine hydroxylase immunopositive neurons would represent anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, if D-SLAM acts to prolong the survival of dopaminergicneurons, it would suggest that D-SLAM may be involved in Parkinson'sDisease.

[0668] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 36 The Effect of D-SLAM on the Growth of Vascular EndothelialCells

[0669] On day 1, human umbilical vein endothelial cells (HUVEC) areseeded at 2-5×10⁴ cells/35 mM dish density in M199 medium containing 4%fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/mlendothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day2, the medium is replaced with M199 containing 10% FBS, 8 units/mlheparin. D-SLAM protein of SEQ ID NO. 2, and positive controls, such asVEGF and basic FGF (bFGF) are added, at varying concentrations. On days4 and 6, the medium is replaced. On day 8, cell number is determinedwith a Coulter Counter.

[0670] An increase in the number of HUVEC cells indicates that D-SLAMmay proliferate vascular endothelial cells.

[0671] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 37 Stimulatory Effect of D-SLAM on the Proliferation of VascularEndothelial Cells

[0672] For evaluation of mitogenic activity of growth factors, thecolorimetric MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) assay with the electron coupling reagent PMS (phenazinemethosulfate) was performed (CellTiter 96 AQ, Promega). Cells are seededin a 96-well plate (5,000 cells/well) in 0.1 ml serum-supplementedmedium and are allowed to attach overnight. After serum-starvation for12 hours in 0.5% FBS, conditions (bFGF, VEGF₁₆₅ or D-SLAM in 0.5% FBS)with or without Heparin (8 U/ml) are added to wells for 48 hours. 20 mgof MTS/PMS mixture (1:0.05) are added per well and allowed to incubatefor 1 hour at 37° C. before measuring the absorbance at 490 nm in anELISA plate reader. Background absorbance from control wells (somemedia, no cells) is subtracted, and seven wells are performed inparallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol.30A:512-518 (1994).

[0673] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 38 Inhibition of PDGF-Induced Vascular Smooth Muscle CellProliferation Stimulatory Effect

[0674] HAoSMC proliferation can be measured, for example, by BrdUrdincorporation. Briefly, subconfluent, quiescent cells grown on the4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then,the cells are pulsed with 10% of serum and 6 mg/ml BrdUrd. After 24 h,immunocytochemistry is performed by using BrdUrd Staining Kit (ZymedLaboratories). In brief, the cells are incubated with the biotinylatedmouse anti-BrdUrd antibody at 4° C. for 2 h after being exposed todenaturing solution and then incubated with the streptavidin-peroxidaseand diaminobenzidine. After counterstaining with hematoxylin, the cellsare mounted for microscopic examination, and the BrdUrd-positive cellsare counted. The BrdUrd index is calculated as a percent of theBrdUrd-positive cells to the total cell number. In addition, thesimultaneous detection of the BrdUrd staining (nucleus) and the FITCuptake (cytoplasm) is performed for individual cells by the concomitantuse of bright field illumination and dark field-UV fluorescentillumination. See, Hayashida et al., J. Biol. Chem.6:271(36):21985-21992 (1996).

[0675] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 39 Stimulation of Endothelial Migration

[0676] This example will be used to explore the possibility that D-SLAMmay stimulate lymphatic endothelial cell migration.

[0677] Endothelial cell migration assays are performed using a 48 wellmicrochemotaxis chamber (Neuroprobe Inc., Cabin John, Md.; Falk, W., etal., J. Immunological Methods 1980;33:239-247).Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um(Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for atleast 6 hours at room temperature and dried under sterile air. Testsubstances are diluted to appropriate concentrations in M199supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of thefinal dilution is placed in the lower chamber of the modified Boydenapparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures arewashed and trypsinized for the minimum time required to achieve celldetachment. After placing the filter between lower and upper chamber,2.5×10⁵ cells suspended in 50 ul M199 containing 1% FBS are seeded inthe upper compartment. The apparatus is then incubated for 5 hours at37° C. in a humidified chamber with 5% CO2 to allow cell migration.After the incubation period, the filter is removed and the upper side ofthe filter with the non-migrated cells is scraped with a rubberpoliceman. The filters are fixed with methanol and stained with a Giemsasolution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration isquantified by counting cells of three random high-power fields (40×) ineach well, and all groups are performed in quadruplicate.

[0678] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 40 Stimulation of Nitric Oxide Production by Endothelial Cells

[0679] Nitric oxide released by the vascular endothelium is believed tobe a mediator of vascular endothelium relaxation. Thus, D-SLAM activitycan be assayed by determining nitric oxide production by endothelialcells in response to D-SLAM.

[0680] Nitric oxide is measured in 96-well plates of confluentmicrovascular endothelial cells after 24 hours starvation and asubsequent 4 hr exposure to various levels of a positive control (suchas VEGF-1) and D-SLAM. Nitric oxide in the medium is determined by useof the Griess reagent to measure total nitrite after reduction of nitricoxide-derived nitrate by nitrate reductase. The effect of D-SLAM onnitic oxide release is examined on HUVEC.

[0681] Briefly, NO release from cultured HUVEC monolayer is measuredwith a NO-specific polarographic electrode connected to a NO meter(Iso-NO, World Precision Instruments Inc.) (1049). Calibration of the NOelements is performed accordingg to the following equation:

2KNO₂+2KI+2H₂SO₄ 6 2NO+I₂+2H₂O+2K₂SO₄

[0682] The standard calibration curve is obtained by adding gradedconcentrations of KNO₂ (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) intothe calibration solution containing KI and H₂SO₄. The specificity of theIso-NO electrode to NO is previously determined by measurement of NOfrom authentic NO gas

[0683] The culture medium is removed and HUVECs are washed twice withDulbecco's phosphate buffered saline. The cells are then bathed in 5 mlof filtered Krebs-Henseleit solution in 6-well plates, and the cellplates are kept on a slide warmer (Lab Line Instruments Inc.) Tomaintain the temperature at 37° C. The NO sensor probe is insertedvertically into the wells, keeping the tip of the electrode 2 mM underthe surface of the solution, before addition of the differentconditions. S-nitroso acetyl penicillamin (SNAP) is used as a positivecontrol. The amount of released NO is expressed as picomoles per 1×10⁶endothelial cells. All values reported are means of four to sixmeasurements in each group (number of cell culture wells). See, Leak etal. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

[0684] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 41 Effect of D-SLAM on Cord Formation in Angiogenesis

[0685] Another step in angiogenesis is cord formation, marked bydifferentiation of endothelial cells. This bioassay measures the abilityof microvascular endothelial cells to form capillary-like structures(hollow structures) when cultured in vitro.

[0686] CADMEC (microvascular endothelial cells) are purchased from CellApplications, Inc. as proliferating (passage 2) cells and are culturedin Cell Applications' CADMEC Growth Medium and used at passage 5. Forthe in vitro angiogenesis assay, the wells of a 48-well cell cultureplate are coated with Cell Applications' Attachment Factor Medium (200ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wellsat 7,500 cells/well and cultured overnight in Growth Medium. The GrowthMedium is then replaced with 300 mg Cell Applications' Chord FormationMedium containing control buffer or D-SLAM (0.1 to 100 ng/ml) and thecells are cultured for an additional 48 hr. The numbers and lengths ofthe capillary-like chords are quantitated through use of the BoeckelerVIA-170 video image analyzer. All assays are done in triplicate.

[0687] Commercial (R&D) VEGF (50 ng/ml) is used as a positive control.b-esteradiol (1 ng/ml) is used as a negative control. The appropriatebuffer (without protein) is also utilized as a control.

[0688] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 42 Angiogenic Effect on Chick Chorioallantoic Membrane

[0689] Chick chorioallantoic membrane (CAM) is a well-established systemto examine angiogenesis. Blood vessel formation on CAM is easily visibleand quantifiable. The ability of D-SLAM to stimulate angiogenesis in CAMcan be examined.

[0690] Fertilized eggs of the White Leghorn chick (Gallus gallus) andthe Japanese qual (Coturnix coturnix) are incubated at 37.8° C. and 80%humidity. Differentiated CAM of 16-day-old chick and 13-day-old qualembryos is studied with the following methods.

[0691] On Day 4 of development, a window is made into the egg shell ofchick eggs. The embryos are checked for normal development and the eggssealed with cellotape. They are further incubated until Day 13.Thermanox coverslips (Nunc, Naperville, Ill.) are cut into disks ofabout 5 mM in diameter. Sterile and salt-free growth factors aredissolved in distilled water and about 3.3 mg/5 ml are pipetted on thedisks. After air-drying, the inverted disks are applied on CAM. After 3days, the specimens are fixed in 3% glutaraldehyde and 2% formaldehydeand rinsed in 0.12 M sodium cacodylate buffer. They are photographedwith a stereo microscope [Wild M8] and embedded for semi- and ultrathinsectioning as described above. Controls are performed with carrier disksalone.

[0692] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 43 Angiogenesis Assay Using a Matrigel Implant in Mouse

[0693] In vivo angiogenesis assay of D-SLAM measures the ability of anexisting capillary network to form new vessels in an implanted capsuleof murine extracellular matrix material (Matrigel). The protein is mixedwith the liquid Matrigel at 4 degree C. and the mixture is then injectedsubcutaneously in mice where it solidifies. After 7 days, the solid“plug” of Matrigel is removed and examined for the presence of new bloodvessels. Matrigel is purchased from Becton DickinsonLabware/Collaborative Biomedical Products.

[0694] When thawed at 4 degree C. the Matrigel material is a liquid. TheMatrigel is mixed with D-SLAM at 150 ng/ml at 4 degree C. and drawn intocold 3 ml syringes. Female C57B1/6 mice approximately 8 weeks old areinjected with the mixture of Matrigel and experimental protein at 2sites at the midventral aspect of the abdomen (0.5 ml/site). After 7days, the mice are sacrificed by cervical dislocation, the Matrigelplugs are removed and cleaned (i.e., all clinging membranes and fibroustissue is removed). Replicate whole plugs are fixed in neutral buffered10% formaldehyde, embedded in paraffin and used to produce sections forhistological examination after staining with Masson's Trichrome. Crosssections from 3 different regions of each plug are processed. Selectedsections are stained for the presence of vWF. The positive control forthis assay is bovine basic FGF (150 ng/ml). Matrigel alone is used todetermine basal levels of angiogenesis.

[0695] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 44 Rescue of Ischemia in Rabbit Lower Limb Model

[0696] To study the in vivo effects of D-SLAM on ischemia, a rabbithindlimb ischemia model is created by surgical removal of one femoralarteries as described previously (Takeshita, S. et al., Am J Pathol147:1649-1660 (1995)). The excision of the femoral artery results inretrograde propagation of thrombus and occlusion of the external iliacartery. Consequently, blood flow to the ischemic limb is dependent uponcollateral vessels originating from the internal iliac artery(Takeshita, S. et al. Am J. Pathol 147:1649-1660 (1995)). An interval of10 days is allowed for post-operative recovery of rabbits anddevelopment of endogenous collateral vessels. At 10 day post-operatively(day 0), after performing a baseline angiogram, the internal iliacartery of the ischemic limb is transfected with 500 mg naked D-SLAMexpression plasmid by arterial gene transfer technology using ahydrogel-coated balloon catheter as described (Riessen, R. et al. HumGene Ther. 4:749-758 (1993); Leclerc, G. et al. J. Clin. Invest. 90:936-944 (1992)). When D-SLAM is used in the treatment, a single bolus of500 mg D-SLAM protein or control is delivered into the internal iliacartery of the ischemic limb over a period of 1 min. through an infusioncatheter. On day 30, various parameters are measured in these rabbits:(a) BP ratio—The blood pressure ratio of systolic pressure of theischemic limb to that of normal limb; (b) Blood Flow and FlowReserve—Resting FL: the blood flow during undilated condition and MaxFL: the blood flow during fully dilated condition (also an indirectmeasure of the blood vessel amount) and Flow Reserve is reflected by theratio of max FL: resting FL; (c) Angiographic Score—This is measured bythe angiogram of collateral vessels. A score is determined by thepercentage of circles in an overlaying grid that with crossing opacifiedarteries divided by the total number m the rabbit thigh; (d) Capillarydensity—The number of collateral capillaries determined in lightmicroscopic sections taken from hindlimbs.

[0697] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 45 Effect of D-SLAM on Vasodilation

[0698] Since dilation of vascular endothelium is important in reducingblood pressure, the ability of D-SLAM to affect the blood pressure inspontaneously hypertensive rats (SHR) is examined. Increasing doses (0,10, 30, 100, 300, and 900 mg/kg) of the D-SLAM are administered to 13-14week old spontaneously hypertensive rats (SHR). Data are expressed asthe mean +/− SEM. Statistical analysis are performed with a pairedt-test and statistical significance is defined as p<0.05 vs. theresponse to buffer alone.

[0699] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 46 Rat Ischemic Skin Flap Model

[0700] The evaluation parameters include skin blood flow, skintemperature, and factor VIII immunohistochemistry or endothelialalkaline phosphatase reaction. D-SLAM expression, during the skinischemia, is studied using in situ hybridization.

[0701] The study in this model is divided into three parts as follows:

[0702] a) Ischemic skin

[0703] b) Ischemic skin wounds

[0704] c) Normal wounds

[0705] The experimental protocol includes:

[0706] d) Raising a 3×4 cm, single pedicle full-thickness random skinflap (myocutaneous flap over the lower back of the animal).

[0707] e) An excisional wounding (4-6 mm in diameter) in the ischemicskin (skin-flap).

[0708] f) Topical treatment with D-SLAM of the excisional wounds (day 0,1, 2, 3, 4 post-wounding) at the following various dosage ranges: 1 mgto 100 mg.

[0709] g) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21post-wounding for histological, immunohistochemical, and in situstudies.

[0710] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 47 Peripheral Arterial Disease Model

[0711] Angiogenic therapy using D-SLAM is a novel therapeutic strategyto obtain restoration of blood flow around the ischemia in case ofperipheral arterial diseases. The experimental protocol includes:

[0712] h) One side of the femoral artery is ligated to create ischemicmuscle of the hindlimb, the other side of hindlimb serves as a control.

[0713] i) b) D-SLAM protein, in a dosage range of 20 mg-500 mg, isdelivered intravenously and/or intramuscularly 3 times (perhaps more)per week for 2-3 weeks.

[0714] j) The ischemic muscle tissue is collected after ligation of thefemoral artery at 1, 2, and 3-weeks for the analysis of D-SLAMexpression and histology. Biopsy is also performed on the other side ofnormal muscle of the contralateral hindlimb.

[0715] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 48 Ischemic Myocardial Disease Model

[0716] D-SLAM is evaluated as a potent mitogen capable of stimulatingthe development of collateral vessels, and restructuring new vesselsafter coronary artery occlusion. Alteration of D-SLAM expression isinvestigated in situ. The experimental protocol includes:

[0717] k) The heart is exposed through a left-side thoracotomy in therat. Immediately, the left coronary artery is occluded with a thinsuture (6-0) and the thorax is closed.

[0718] l) D-SLAM protein, in a dosage range of 20 mg -500 mg, isdelivered intravenously and/or intramuscularly 3 times (perhaps more)per week for 2-4 weeks.

[0719] m) Thirty days after the surgery, the heart is removed andcross-sectioned for morphometric and in situ analyzes.

[0720] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 49 Rat Corneal Wound Healing Model

[0721] This animal model shows the effect of D-SLAM onneovascularization. The experimental protocol includes:

[0722] a) Making a 1-1.5 mm long incision from the center of cornea intothe stromal layer.

[0723] b) Inserting a spatula below the lip of the incision facing theouter corner of the eye.

[0724] c) Making a pocket (its base is 1-1.5 mm form the edge of theeye).

[0725] d) Positioning a pellet, containing 50 ng-5 ug of D-SLAM, withinthe pocket.

[0726] e) D-SLAM treatment can also be applied topically to the cornealwounds in a dosage range of 20 mg -500 mg (daily treatment for fivedays).

[0727] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 50 Diabetic Mouse and Glucocorticoid-Impaired Wound HealingModels

[0728] A. Diabetic db+/db+ Mouse Model

[0729] To demonstrate that D-SLAM accelerates the healing process, thegenetically diabetic mouse model of wound healing is used. The fullthickness wound healing model in the db+/db+ mouse is a wellcharacterized, clinically relevant and reproducible model of impairedwound healing. Healing of the diabetic wound is dependent on formationof granulation tissue and re-epithelialization rather than contraction(Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G.et al., Am. J. Pathol. 136:1235 (1990)).

[0730] The diabetic animals have many of the characteristic featuresobserved in Type II diabetes mellitus. Homozygous (db+/db+) mice areobese in comparison to their normal heterozygous (db+/+m) littermates.Mutant diabetic (db+/db+) mice have a single autosomal recessivemutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci.USA 77:283-293 (1982)). Animals show polyphagia, polydipsia andpolyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose,increased or normal insulin levels, and suppressed cell-mediatedimmunity (Mandel et al., J. Immunol. 120:1375 (1978); Debray-Sachs, M.et al., Clin. Exp. Immunol. 51(1):1-7 (1983); Leiter et al., Am. J. ofPathol. 114:46-55 (1985)). Peripheral neuropathy, myocardialcomplications, and microvascular lesions, basement membrane thickeningand glomerular filtration abnormalities have been described in theseanimals (Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertsonet al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest.40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl):1-6 (1982)).These homozygous diabetic mice develop hyperglycemia that is resistantto insulin analogous to human type II diabetes (Mandel et al., J.Immunol. 120:1375-1377 (1978)).

[0731] The characteristics observed in these animals suggests thathealing in this model may be similar to the healing observed in humandiabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

[0732] Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andare 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Human Genome Sciences, Inc.Institutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

[0733] Wounding protocol is performed according to previously reportedmethods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mM full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

[0734] Wounds are visually examined and photographed at a fixed distanceat the day of surgery and at two day intervals thereafter. Wound closureis determined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

[0735] D-SLAM is administered using at a range different doses ofD-SLAM, from 4 mg to 500 mg per wound per day for 8 days in vehicle.Vehicle control groups received 50 mL of vehicle solution.

[0736] Animals are euthanized on day 8 with an intraperitoneal injectionof sodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

[0737] Three groups of 10 animals each (5 diabetic and 5 non-diabeticcontrols) are evaluated: 1) Vehicle placebo control, 2) D-SLAM.

[0738] Wound closure is analyzed by measuring the area in the verticaland horizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 is 64 mm², the corresponding size of the dermalpunch. Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

[0739] Specimens are fixed in 10% buffered formalin and paraffinembedded blocks are sectioned perpendicular to the wound surface (5 mM)and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment with D-SLAM. This assessment included verification of thepresence of cell accumulation, inflammatory cells, capillaries,fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D.G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometeris used by a blinded observer.

[0740] Tissue sections are also stained immunohistochemically with apolyclonal rabbit anti-human keratin antibody using ABC Elite detectionsystem. Human skin is used as a positive tissue control while non-immuneIgG is used as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

[0741] Proliferating cell nuclear antigen/cyclin (PCNA) in skinspecimens is demonstrated by using anti-PCNA antibody (1:50) with an ABCElite detection system. Human colon cancer served as a positive tissuecontrol and human brain tissue is used as a negative tissue control.Each specimen included a section with omission of the primary antibodyand substitution with non-immune mouse IgG. Ranking of these sections isbased on the extent of proliferation on a scale of 0-8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

[0742] Experimental data are analyzed using an unpaired t test. A pvalue of <0.05 is considered significant.

[0743] B. Steroid Impaired Rat Model

[0744] The inhibition of wound healing by steroids has been welldocumented in various in vitro and in vivo systems (Wahl, S. M.Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid Action:Basic and Clinical Aspects. 280-302 (1989); Wahl, S. M.et al., J.Immunol. 115: 476-481 (1975); Werb, Z. et al., J. Exp. Med.147:1684-1694 (1978)). Glucocorticoids retard wound healing byinhibiting angiogenesis, decreasing vascular permeability (Ebert, R. H.,et al., An. Intern. Med. 37:701-705 (1952)), fibroblast proliferation,and collagen synthesis (Beck, L. S. et al., Growth Factors. 5: 295-304(1991); Haynes, B. F. et al., J. Clin. Invest. 61: 703-797 (1978)) andproducing a transient reduction of circulating monocytes (Haynes, B. F.,et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, S. M.,“Glucocorticoids and wound healing”, In: Antiinflammatory SteroidAction: Basic and Clinical Aspects, Academic Press, New York, pp.280-302 (1989)). The systemic administration of steroids to impairedwound healing is a well establish phenomenon in rats (Beck, L. S. etal., Growth Factors. 5: 295-304 (1991); Haynes, B. F., et al., J. Clin.Invest. 61: 703-797 (1978); Wahl, S. M., “Glucocorticoids and woundhealing”, In: Antiinflammatory Steroid Action: Basic and ClinicalAspects, Academic Press, New York, pp. 280-302 (1989); Pierce, G. F. etal., Proc. Natl. Acad. Sci. USA 86: 2229-2233 (1989)).

[0745] To demonstrate that D-SLAM can accelerate the healing process,the effects of multiple topical applications of D-SLAM on full thicknessexcisional skin wounds in rats in which healing has been impaired by thesystemic administration of methylprednisolone is assessed.

[0746] Young adult male Sprague Dawley rats weighing 250-300 g (CharlesRiver Laboratories) are used in this example. The animals are purchasedat 8 weeks of age and are 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy is conducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

[0747] The wounding protocol is followed according to section A, above.On the day of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

[0748] Wounds are visually examined and photographed at a fixed distanceat the day of wounding and at the end of treatment. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

[0749] D-SLAM is administered using at a range different doses ofD-SLAM, from 4 mg to 500 mg per wound per day for 8 days in vehicle.Vehicle control groups received 50 mL of vehicle solution.

[0750] Animals are euthanized on day 8 with an intraperitoneal injectionof sodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

[0751] Four groups of 10 animals each (5 with methylprednisolone and 5without glucocorticoid) are evaluated: 1) Untreated group 2) Vehicleplacebo control 3) D-SLAM treated groups.

[0752] Wound closure is analyzed by measuring the area in the verticaland horizontal axis and obtaining the total area of the wound. Closureis then estimated by establishing the differences between the initialwound area (day 0) and that of post treatment (day 8). The wound area onday 1 is 64 mm², the corresponding size of the dermal punch.Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

[0753] Specimens are fixed in 10% buffered formalin and paraffinembedded blocks are sectioned perpendicular to the wound surface (5 mm)and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds allows assessment of whether the healingprocess and the morphologic appearance of the repaired skin is improvedby treatment with D-SLAM. A calibrated lens micrometer is used by ablinded observer to determine the distance of the wound gap.

[0754] Experimental data are analyzed using an unpaired t test. A pvalue of <0.05 is considered significant.

[0755] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 51 Lymphadema Animal Model

[0756] The purpose of this experimental approach is to create anappropriate and consistent lymphedema model for testing the therapeuticeffects of D-SLAM in lymphangiogenesis and re-establishment of thelymphatic circulatory system in the rat hind limb. Effectiveness ismeasured by swelling volume of the affected limb, quantification of theamount of lymphatic vasculature, total blood plasma protein, andhistopathology. Acute lymphedema is observed for 7-10 days. Perhaps moreimportantly, the chronic progress of the edema is followed for up to 3-4weeks.

[0757] Prior to beginning surgery, blood sample is drawn for proteinconcentration analysis. Male rats weighing approximately ˜350 g aredosed with Pentobarbital. Subsequently, the right legs are shaved fromknee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH.Blood is drawn for serum total protein testing. Circumference andvolumetric measurements are made prior to injecting dye into paws aftermarking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsalpaw). The intradermal dorsum of both right and left paws are injectedwith 0.05 ml of 1% Evan's Blue. Circumference and volumetricmeasurements are then made following injection of dye into paws.

[0758] Using the knee joint as a landmark, a mid-leg inguinal incisionis made circumferentially allowing the femoral vessels to be located.Forceps and hemostats are used to dissect and separate the skin flaps.After locating the femoral vessels, the lymphatic vessel that runs alongside and underneath the vessel(s) is located. The main lymphatic vesselsin this area are then electrically coagulated or suture ligated.

[0759] Using a microscope, muscles in back of the leg (near thesemitendinosis and adductors) are bluntly dissected. The popliteal lymphnode is then located. The 2 proximal and 2 distal lymphatic vessels anddistal blood supply of the popliteal node are then and ligated bysuturing. The popliteal lymph node, and any accompanying adipose tissue,is then removed by cutting connective tissues.

[0760] Care is taken to control any mild bleeding resulting from thisprocedure. After lymphatics are occluded, the skin flaps are sealed byusing liquid skin (Vetbond) (A J Buck). The separated skin edges aresealed to the underlying muscle tissue while leaving a gap of ˜0.5 cmaround the leg. Skin also may be anchored by suturing to underlyingmuscle when necessary.

[0761] To avoid infection, animals are housed individually with mesh (nobedding). Recovering animals are checked daily through the optimaledematous peak, which typically occurred by day 5-7. The plateauedematous peak are then observed. To evaluate the intensity of thelymphedema, the circumference and volumes of 2 designated places on eachpaw before operation and daily for 7 days are measured. The effectplasma proteins on lymphedema is determined and whether protein analysisis a useful testing perimeter is also investigated. The weights of bothcontrol and edematous limbs are evaluated at 2 places. Analysis isperformed in a blind manner.

[0762] Circumference Measurements:

[0763] Under brief gas anesthetic to prevent limb movement, a cloth tapeis used to measure limb circumference. Measurements are done at theankle bone and dorsal paw by 2 different people then those 2 readingsare averaged. Readings are taken from both control and edematous limbs.

[0764] Volumetric Measurements:

[0765] On the day of surgery, animals are anesthetized withPentobarbital and are tested prior to surgery. For daily volumetricsanimals are under brief halothane anesthetic (rapid immobilization andquick recovery), both legs are shaved and equally marked usingwaterproof marker on legs. Legs are first dipped in water, then dippedinto instrument to each marked level then measured by Buxco edemasoftware (Chen/Victor). Data is recorded by one person, while the otheris dipping the limb to marked area.

[0766] Blood-Plasma Protein Measurements:

[0767] Blood is drawn, spun, and serum separated prior to surgery andthen at conclusion for total protein and Ca2+ comparison.

[0768] Limb Weight Comparison:

[0769] After drawing blood, the animal is prepared for tissuecollection. The limbs are amputated using a quillitine, then bothexperimental and control legs are cut at the ligature and weighed. Asecond weighing is done as the tibio-cacaneal joint is disarticulatedand the foot is weighed.

[0770] Histological Preparations:

[0771] The transverse muscle located behind the knee (popliteal) area isdissected and arranged in a metal mold, filled with freezegel, dippedinto cold methylbutane, placed into labeled sample bags at −80EC untilsectioning. Upon sectioning, the muscle is observed under fluorescentmicroscopy for lymphatics.

[0772] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

Example 52 Suppression of TNF Alpha-Induced Adhesion Molecule Expressionby D-SLAM

[0773] The recruitment of lymphocytes to areas of inflammation andangiogenesis involves specific receptor-ligand interactions between cellsurface adhesion molecules (CAMs) on lymphocytes and the vascularendothelium. The adhesion process, in both normal and pathologicalsettings, follows a multi-step cascade that involves intercellularadhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1(VCAM-1), and endothelial leukocyte adhesion molecule-I (E-selectin)expression on endothelial cells (EC). The expression of these moleculesand others on the vascular endothelium determines the efficiency withwhich leukocytes may adhere to the local vasculature and extravasateinto the local tissue during the development of an inflammatoryresponse. The local concentration of cytokines and growth factorparticipate in the modulation of the expression of these CAMs.

[0774] Tumor necrosis factor alpha (TNF-a), a potent proinflammatorycytokine, is a stimulator of all three CAMs on endothelial cells and maybe involved in a wide variety of inflammatory responses, often resultingin a pathological outcome.

[0775] The potential of D-SLAM to mediate a suppression of TNF-a inducedCAM expression can be examined. A modified ELISA assay which uses ECs asa solid phase absorbent is employed to measure the amount of CAMexpression on TNF-a treated ECs when co-stimulated with a member of theFGF family of proteins.

[0776] To perform the experiment, human umbilical vein endothelial cell(HUVEC) cultures are obtained from pooled cord harvests and maintainedin growth medium (EGM-2; Clonetics, San Diego, Calif.) supplemented with10% FCS and 1% penicillin/streptomycin in a 37 degree C. humidifiedincubator containing 5% CO₂. HUVECs are seeded in 96-well plates atconcentrations of 1×10⁴ cells/well in EGM medium at 37 degree C. for18-24 hrs or until confluent. The monolayers are subsequently washed 3times with a serum-free solution of RPMI-1640 supplemented with 100 U/mlpenicillin and 100 mg/ml streptomycin, and treated with a given cytokineand/or growth factor(s) for 24 h at 37 degree C. Following incubation,the cells are then evaluated for CAM expression.

[0777] Human Umbilical Vein Endothelial cells (HUVECs) are grown in astandard 96 well plate to confluence. Growth medium is removed from thecells and replaced with 90 ul of 199 Medium (10% FBS). Samples fortesting and positive or negative controls are added to the plate intriplicate (in 10 ul volumes). Plates are incubated at 37 degree C. foreither 5 h (selectin and integrin expression) or 24 h (integrinexpression only). Plates are aspirated to remove medium and 100 μl of0.1% paraformaldehyde-PBS (with Ca++ and Mg++) is added to each well.Plates are held at 4° C. for 30 min.

[0778] Fixative is then removed from the wells and wells are washed 1×with PBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry.Add 10 μl of diluted primary antibody to the test and control wells.Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin areused at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stockantibody). Cells are incubated at 37° C. for 30 min. in a humidifiedenvironment. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA.

[0779] Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphotase(1:5,000 dilution) to each well and incubated at 37° C. for 30 min.Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-NitrophenolPhosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μlof pNPP substrate in glycine buffer is added to each test well. Standardwells in triplicate are prepared from the working dilution of theExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000(10⁰)>10^(−0.5)>10⁻¹>10^(−1.5)0.5 μl of each dilution is added totriplicate wells and the resulting AP content in each well is 5.50 ng,1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added toeach of the standard wells. The plate must be incubated at 37° C. for 4h. A volume of 50 μl of 3M NaOH is added to all wells. The results arequantified on a plate reader at 405 nm. The background subtractionoption is used on blank wells filled with glycine buffer only. Thetemplate is set up to indicate the concentration of AP-conjugate in eachstandard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results areindicated as amount of bound AP-conjugate in each sample.

[0780] The studies described in this example tested activity in D-SLAMprotein. However, one skilled in the art could easily modify theexemplified studies to test the activity of D-SLAM polynucleotides(e.g., gene therapy), agonists, and/or antagonists of D-SLAM.

[0781] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples. Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0782] The entire disclosure of each document cited (including patents,patent applications, journal articles, abstracts, laboratory manuals,books, or other disclosures) in the Background of the Invention,Detailed Description, and Examples is hereby incorporated herein byreference. Moreover, the sequence listing is herein incorporated byreference.

1 13 1 3220 DNA Homo sapiens 1 gaaggaccac agctcctccc gtgcatccactcggcctggg aggttctgga ttttggctgt 60 cgagggagtt tgcctgcctc tccagagaaagatggtcatg aggcccctgt ggagtctgct 120 tctctgggaa gccctacttc ccattacagttactggtgcc caagtgctga gcaaagtcgg 180 gggctcggtg ctgctggtgg cagcgcgtccccctggcttc caagtccgtg aggctatctg 240 gcgatctctc tggccttcag aagagctcctggccacgttt ttccgaggct ccctggagac 300 tctgtaccat tcccgcttcc tgggccgagcccagctacac agcaacctca gcctggagct 360 cgggccgctg gagtctggag acagcggcaacttctccgtg ttgatggtgg acacaagggg 420 ccagccctgg acccagaccc tccagctcaaggtgtacgat gcagtgccca ggcccgtggt 480 acaagtgttc attgctgtag aaagggatgctcagccctcc aagacctgcc aggttttctt 540 gtcctgttgg gcccccaaca tcagcgaaataacctatagc tggcgacggg agacaaccat 600 ggactttggt atggaaccac acagcctcttcacagacgga caggtgctga gcatttccct 660 gggaccagga gacagagatg tggcctattcctgcattgtc tccaaccctg tcagctggga 720 cttggccaca gtcacgccct gggatagctgtcatcatgag gcagcaccag ggaaggcctc 780 ctacaaagat gtgctgctgg tggtggtgcctgtctcgctg ctcctgatgc tggttactct 840 cttctctgcc tggcactggt gcccctgctcagggaaaaag aaaaaggatg tccatgctga 900 cagagtgggt ccagagacag agaacccccttgtgcaggat ctgccataaa ggacaatatg 960 aactgatgcc tggactatca gtaaccccactgcacaggca cacgatgctc tgggacataa 1020 ctggtgcctg gaaatcacca tggtcctcatatctcccatg ggaatcctgt cctgcctcga 1080 aggagcagcc tgggcagcca tcacaccacgaggacaggaa gcaccagcac gtttcacacc 1140 tcccccttcc ctctcccatc ttctcatatcctggctcttc tctgggcaag atgagccaag 1200 cagaacattc catccaggac actggaagttctccaggatc cagatccatg gggacattaa 1260 tagtccaagg cattccctcc cccaccactattcataaagt attaaccaac tggcaccaag 1320 gaattgcctc cagcctgagt cctaggctctaaaagatatt acatatttga actaatagag 1380 gaactctgag tcacccatgc cagcatcagcttcagcccca gaccctgcag tttgagatct 1440 gatgcttcct gagggccaag gcattgctgtaagaaaaggt ctagaaatag gtgaaagtga 1500 gaggtggggg acaggggttt ctctttctggcctaaggact ttcaggtaat cagagttcat 1560 gggccctcaa aggtaaattg cagttgtagacaccgaggat ggttgacaac ccatggttga 1620 gatgggcacc gttttgcagg aaacaccatattaatagaca tcctcaccat ctccatccgc 1680 tctcacgcct cctgcaggat ctgggagtgagggtggagag tctttcctca cgctccagca 1740 cagtggccag gaaaagaaat actgaatttgccccagccaa caggacgttc ttgcacaact 1800 tcaagaaaag cagctcagct caggatgagtcttcctgcct gaaactgaga gagtgaagaa 1860 ccataaaacg ctatgcagaa ggaacattatggagagaaag ggtactgagg cactctagaa 1920 tctgccacat tcattttcaa atgcaaatgcagaagactta ccttagttca aggggagggg 1980 acaaagaccc cacagcccaa cagcaggactgtagaggtca ctctgactcc atcaaacttt 2040 ttattgtggc catcttagga aaatacattctgcccctgaa tgattctgtc tagaaaagct 2100 ctggagtatt gatcactact ggaaaaacacttaaggagct aaacttacct tcggggatta 2160 ttagctgata aggttcacag tttctctcacccaggtgtaa ctggattttt tctggggcct 2220 caatccagtc ttgataacag cgaggaaagaggtattgaag aaacaggggt gggtttgaag 2280 tactattttc cccagggtgg cttcaatctccccacctagg atgtcagccc tgtccaagga 2340 ccttccctct tctcccccag ttccctgggcaatcacttca ccttggacaa aggatcagca 2400 cagctggcct ccagatccac atcaccactcttccactcga ttgttcccag atcctccctg 2460 cctggcctgc tcagaggttc cctgttggtaacctggcttt atcaaattct catccctttc 2520 ccacacccac ttctctccta tcaccttcccccaagattac ctgaacaggg tccatggcca 2580 ctcaacctgt cagcttgcac catccccacctgccacctac agtcaggcca catgcctggt 2640 cactgaatca tgcaaaactg gcctcagtccctaaaaatga tgtggaaagg aaagcccagg 2700 atctgacaat gagccctggt ggatttgtggggaaaaaata cacagcactc cccacctttc 2760 tttcgttcat ctccagggcc ccacctcagatcaaagcagc tctggatgag atgggacctg 2820 cagctctccc tccacaaggt gactcttagcaacctcattt cgacagtggt ttgtagcgtg 2880 gtgcaccagg gccttgttga acagatccacactgctctaa taaagttccc atccttaatg 2940 actcacttgt caactagtgg actaattaaccctccaccaa aaaaacacaa agtgcttctg 3000 tgagaccaat tttgtgctaa tgagcattgagactgatgct ttgtaagtca caccacaaca 3060 aatattgatt gagggcgctg catgtgctgggtacatttct tggcacttgg gaatcagtag 3120 tcaagcgaaa cccttgcctt tgagagtttatggtctggat aatataaata aacaagtaag 3180 cataaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa 3220 2 285 PRT Homo sapiens 2 Met Val Met Arg Pro Leu Trp SerLeu Leu Leu Trp Glu Ala Leu Leu 1 5 10 15 Pro Ile Thr Val Thr Gly AlaGln Val Leu Ser Lys Val Gly Gly Ser 20 25 30 Val Leu Leu Val Ala Ala ArgPro Pro Gly Phe Gln Val Arg Glu Ala 35 40 45 Ile Trp Arg Ser Leu Trp ProSer Glu Glu Leu Leu Ala Thr Phe Phe 50 55 60 Arg Gly Ser Leu Glu Thr LeuTyr His Ser Arg Phe Leu Gly Arg Ala 65 70 75 80 Gln Leu His Ser Asn LeuSer Leu Glu Leu Gly Pro Leu Glu Ser Gly 85 90 95 Asp Ser Gly Asn Phe SerVal Leu Met Val Asp Thr Arg Gly Gln Pro 100 105 110 Trp Thr Gln Thr LeuGln Leu Lys Val Tyr Asp Ala Val Pro Arg Pro 115 120 125 Val Val Gln ValPhe Ile Ala Val Glu Arg Asp Ala Gln Pro Ser Lys 130 135 140 Thr Cys GlnVal Phe Leu Ser Cys Trp Ala Pro Asn Ile Ser Glu Ile 145 150 155 160 ThrTyr Ser Trp Arg Arg Glu Thr Thr Met Asp Phe Gly Met Glu Pro 165 170 175His Ser Leu Phe Thr Asp Gly Gln Val Leu Ser Ile Ser Leu Gly Pro 180 185190 Gly Asp Arg Asp Val Ala Tyr Ser Cys Ile Val Ser Asn Pro Val Ser 195200 205 Trp Asp Leu Ala Thr Val Thr Pro Trp Asp Ser Cys His His Glu Ala210 215 220 Ala Pro Gly Lys Ala Ser Tyr Lys Asp Val Leu Leu Val Val ValPro 225 230 235 240 Val Ser Leu Leu Leu Met Leu Val Thr Leu Phe Ser AlaTrp His Trp 245 250 255 Cys Pro Cys Ser Gly Lys Lys Lys Lys Asp Val HisAla Asp Arg Val 260 265 270 Gly Pro Glu Thr Glu Asn Pro Leu Val Gln AspLeu Pro 275 280 285 3 335 PRT Homo sapiens 3 Met Asp Pro Lys Gly Leu LeuSer Leu Thr Phe Val Leu Phe Leu Ser 1 5 10 15 Leu Ala Phe Gly Ala SerTyr Gly Thr Gly Gly Arg Met Met Asn Cys 20 25 30 Pro Lys Ile Leu Arg GlnLeu Gly Ser Lys Val Leu Leu Pro Leu Thr 35 40 45 Tyr Glu Arg Ile Asn LysSer Met Asn Lys Ser Ile His Ile Val Val 50 55 60 Thr Met Ala Lys Ser LeuGlu Asn Ser Val Glu Asn Lys Ile Val Ser 65 70 75 80 Leu Asp Pro Ser GluAla Gly Pro Pro Arg Tyr Leu Gly Asp Arg Tyr 85 90 95 Lys Phe Tyr Leu GluAsn Leu Thr Leu Gly Ile Arg Glu Ser Arg Lys 100 105 110 Glu Asp Glu GlyTrp Tyr Leu Met Thr Leu Glu Lys Asn Val Ser Val 115 120 125 Gln Arg PheCys Leu Gln Leu Arg Leu Tyr Glu Gln Val Ser Thr Pro 130 135 140 Glu IleLys Val Leu Asn Lys Thr Gln Glu Asn Gly Thr Cys Thr Leu 145 150 155 160Ile Leu Gly Cys Thr Val Glu Lys Gly Asp His Val Ala Tyr Ser Trp 165 170175 Ser Glu Lys Ala Gly Thr His Pro Leu Asn Pro Ala Asn Ser Ser His 180185 190 Leu Leu Ser Leu Thr Leu Gly Pro Gln His Ala Asp Asn Ile Tyr Ile195 200 205 Cys Thr Val Ser Asn Pro Ile Ser Asn Asn Ser Gln Thr Phe SerPro 210 215 220 Trp Pro Gly Cys Arg Thr Asp Pro Ser Glu Thr Lys Pro TrpAla Val 225 230 235 240 Tyr Ala Gly Leu Leu Gly Gly Val Ile Met Ile LeuIle Met Val Val 245 250 255 Ile Leu Gln Leu Arg Arg Arg Gly Lys Thr AsnHis Tyr Gln Thr Thr 260 265 270 Val Glu Lys Lys Ser Leu Thr Ile Tyr AlaGln Val Gln Lys Pro Gly 275 280 285 Pro Leu Gln Lys Lys Leu Asp Ser PhePro Ala Gln Asp Pro Cys Thr 290 295 300 Thr Ile Tyr Val Ala Ala Thr GluPro Val Pro Glu Ser Val Gln Glu 305 310 315 320 Thr Asn Ser Ile Thr ValTyr Ala Ser Val Thr Leu Pro Glu Ser 325 330 335 4 733 DNA Homo sapiens 4gggatccgga gcccaaatct tctgacaaaa ctcacacatg cccaccgtgc ccagcacctg 60aattcgaggg tgcaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga 120tctcccggac tcctgaggtc acatgcgtgg tggtggacgt aagccacgaa gaccctgagg 180tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg 240aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact 300ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca acccccatcg 360agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc 420catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct 480atccaagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga 540ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg 600acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc 660acaaccacta cacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccgc 720gactctagag gat 733 5 5 PRT Homo sapiens MISC_FEATURE (3) Xaa equals anyamino acid 5 Trp Ser Xaa Trp Ser 1 5 6 86 DNA Homo sapiens 6 gcgcctcgagatttccccga aatctagatt tccccgaaat gatttccccg aaatgatttc 60 cccgaaatatctgccatctc aattag 86 7 27 DNA Homo sapiens 7 gcggcaagct ttttgcaaagcctaggc 27 8 271 DNA Homo sapiens 8 ctcgagattt ccccgaaatc tagatttccccgaaatgatt tccccgaaat gatttccccg 60 aaatatctgc catctcaatt agtcagcaaccatagtcccg cccctaactc cgcccatccc 120 gcccctaact ccgcccagtt ccgcccattctccgccccat ggctgactaa ttttttttat 180 ttatgcagag gccgaggccg cctcggcctctgagctattc cagaagtagt gaggaggctt 240 ttttggaggc ctaggctttt gcaaaaagct t271 9 32 DNA Homo sapiens 9 gcgctcgagg gatgacagcg atagaacccc gg 32 10 31DNA Homo sapiens 10 gcgaagcttc gcgactcccc ggatccgcct c 31 11 12 DNA Homosapiens 11 ggggactttc cc 12 12 73 DNA Homo sapiens 12 gcggcctcgaggggactttc ccggggactt tccggggact ttccgggact ttccatcctg 60 ccatctcaat tag73 13 256 DNA Homo sapiens 13 ctcgagggga ctttcccggg gactttccggggactttccg ggactttcca tctgccatct 60 caattagtca gcaaccatag tcccgcccctaactccgccc atcccgcccc taactccgcc 120 cagttccgcc cattctccgc cccatggctgactaattttt tttatttatg cagaggccga 180 ggccgcctcg gcctctgagc tattccagaagtagtgagga ggcttttttg gaggcctagg 240 cttttgcaaa aagctt 256

What is claimed is
 1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence from the group consisiting of: (a) a polynucleotide fragment of SEQ ID NO:1 or a polynucleotide fragment of the cDNA sequence included in ATCC Deposit No: 209623; (b) a polynuclotide encoding a polypeptide fragment of SEQ ID NO:2 or the cDNA sequence included in ATCC Deposit No: 209623; (c) a polynucleotide encoding a polypeptide domain of SEQ ID NO:2 or the cDNA sequence included in ATCC Deposit No: 209623; (d) a polynucleotide encoding a polypeptide epitope of SEQ ID NO:2 or cDNA sequence included in ATCC Deposit No: 209623; (e) a polynucleotide encoding a polypeptide of SEQ ID NO:2 or the cDNA sequence included in ATCC Deposit No: 209623 having biological activity; (f) a polynucleotide which is a variant of SEQ ID NO:1; (g) a polynucleotide which is an allelic variant of SEQ ID NO:1; (h) a polynucleotide which encodes a species homologue of the SEQ ID NO:2; (i) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h), wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues.
 2. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a mature form or a secreted protein.
 3. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO:2 or the coding sequence included in ATCC Deposit No:
 209623. 4. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO:1 or the cDNA sequence included in ATCC Deposit No:
 209623. 5. The isolated nucleic acid molecule of claim 2, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.
 6. The isolated nucleic acid molecule of claim 3, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.
 7. A recombinant vector comprising the isolated nucleic acid molecule of claim
 1. 8. A method of makin a recombinant host cell comprising the isolated nucleic acid molecule of claim
 1. 9. A recombinant host cell produced by the method of claim
 9. 10. The recombinant host cell of claim 9 comprising vector sequences.
 11. An isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of: (a) a polypeptide fragment of SEQ ID NO:2 or the encoded sequence included in ATCC Deposit No: 209623; (b) a polypeptide fragment of SEG ID NO:2 or the encoded sequence included in ATCC Deposit No: 209623 having biological activity; (c) a polypeptide domain of SEQ ID NO:2 or the encoded sequence included in ATCC Deposit No: 209623; (d) a polypeptide epitope of SEQ ID NO:2 or the encoded sequence included in ATCC Deposit No: 209623; (e) a mature form of a secreted protein; (f) a full length secreted protein; (g) a variant of SEQ ID NO:2 (h) an allelic variant of SEQ ID NO:2; or (i) a species homologue of the SEQ ID NO:2.
 12. The isolated polypeptide of claim 11, wherein the mature form or the full length secreted protein comprises sequential acid deletions from either the C-terminus or the N-terminus.
 13. An isolated antibody that binds specifically to the isolated polypeptide of claim
 11. 14. A recombinant host cell that expressed the isolated polypeptide of claim
 11. 15. A method of making an isolated polypeptide comprising: (a) culturing the recombinant host cell of claim 14 under conditions such that said polypeptide is expressed; and (b) recovering said polypeptide.
 16. The polypeptide produced by claim
 15. 17. A method for preveting, treating, or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of the polypeptide of claim
 11. 18. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject related to expression or activity of a secreted protein comprising: (a) determining the presence or absence of a mutation in the polynucleotide of claim 1; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based ont he presence or absence of said mutation.
 19. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject related to expression or activitu of a secreted protein comprising: (a) determining the presence or amount of expression of the polypeptides of claim 11 in a biological sample; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.
 20. A method for identifying binding partner to the polypeptide of claim 11 comprising: (a) contacting the polypeptide of claim 11 with a binding partner; and (b) determining whether the binding partner effects an activity of the polypeptide.
 21. The gene corresponding to the cDNA sequence of SEQ ID NO:2.
 22. A method of identifying an activity in a biological assay, wherein the method comprises: (a) expressing SEQ ID NO:1 in a cell; (b) isolating the supernatant; (c) detecting an activity in a biological assay; and (d) identifying the protein in the supernatant having the activity.
 23. The product produced by the method of claim
 22. 